Joint TDR/EC Expert Consultation on Biomarkers in Tuberculosis

Special Programme for Research & Training in Tropical Diseases (TDR) sponsored by UNICEF/UNDP/World Bank/WHO

Joint TDR/EC expert consultation on biomarkers in tuberculosis

Report of the joint TDR/EC expert consultation to evaluate the potential roles of biomarkers in the management of HIV-infected and HIV-uninfected patients with tuberculosis

Geneva, Switzerland, 2–3 July 2008

Special Programme for Research & Training in Tropical Diseases (TDR) sponsored by UNICEF/UNDP/World Bank/WHO

Joint TDR/EC expert consultation on biomarkers in tuberculosis

Report of the joint TDR/EC expert consultation to evaluate the potential roles of biomarkers in the management of HIV-infected and HIV-uninfected patients with tuberculosis

Geneva, Switzerland, 2–3 July 2008

Authors: Professor Alimuddin Zumla, Dr Robert Wallis, Professor Mark Doherty, Professor Nigel Klein, Dr Shreemanta Parida, Dr Ole Olesen, Dr Hannu Lång, Dr Mahnaz Vahedi and Dr Philip Onyebujoh

Final Biomarker report.indd 1 09.04.09 12:57 WHO Library Cataloguing-in-Publication Data

Joint TDR/EC expert consultation on biomarkers in tuberculosis: report of the joint TDR/EC expert consultation to evaluate the potential roles of biomarkers in the management of HIV-infected and HIV-uninfected patients with tuberculosis, Geneva, Switzerland, 2–3 July 2008 / Alimuddin Zumla … [et al].

1.Tuberculosis, Multidrug-resistant - drug therapy. 2.Extensively drug-resistant tuberculosis - drug therapy. 3.Biological markers - analysis. 4.Tuberculosis vaccines - immunology. 5.HIV infections - complications. I.Zumla, Alimuddin. II.Wallis, Robert. III.Klein, Nigel. IV.Olesen, Ole. V.Lång, Hannu. VI.Vahedi, Mahnaz. VII.Onyebujoh, Philip. VIII.UNICEF/UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

ISBN 978 92 4 159794 4 (NLM classification: WF 200)

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Final Biomarker report.indd 2 09.04.09 12:57 Final Biomarker report.indd 3 6. Candidatehostmarkers summary Executive 5. Candidatemicrobiologicalmarkers 4. Clinicalneedsformarkers 3. TheTBandHIVpandemics:coinfectionissues 2. Idealcharacteristicsofmarkersandsurrogateend-points 1. Introduction Abbreviations 6.11. 6.10. 6.9. 6.8. 6.7. 6.5. 6.4. 6.3. 6.2. 6.1. 5.5. 5.4. 5.3. 6.6. 4.1. 5.2. 5.1. 4.4. 4.3. 4.2.

Further researchonhostimmunemarkers Research questionsarising Use ofsetsmarkerstoincreasethepredictivevalue Whole bloodkillingassays IL-4/IL-4δ2 ratio Host proteins 6.5.3. Solubleurokinaseplasminogenactivatorreceptor(suPAR) 6.5.2. Erythrocytesedimentationrate(ESR)andC-reactiveprotein (CRP) 6.5.1. Neopterin Other nonspecificimmuneparametersaspredictorsoftreatmentoutcome Soluble intercellularadhesionmoleculetype1(sICAM-1) Host nonspecificimmuneparametersasbiomarkersfortreatmentresponse Antibodies tomycobacterialenzymesand38kDaantigen Interferon-gamma releaseassays(IGRAs) Volatile mycobacterialmarkersinbreath 5.4.2. UrinemycobacterialLAM 5.4.1. Trans-renal mycobacterialDNA Mycobacterial markersinurine Mycobacterial antigens:antigen85and85B 5.2.1. Quantitativesputummicrobiologicalstudies Mycobacterial load The basisandplausibilityofbacteriologicalbiomarkers Approaches tostudyingbiomarkers Biomarkers oftreatmentoutcome Biomarkers oftreatmenteffect Biomarkers ofextentdisease ...... 7 ...... 25 ...... 9 ...... 25 ...... 19 ...... 13 ...... 27 ...... 26 ...... 22 ...... 14 ...... 22 ...... 13 ...... 18 ...... 14 ...... 15 ...... 21 Contents ...... 22 ...... 28 ...... 23 ...... 13 ...... 21 ...... 21 ...... 26 ...... 26 ...... 18 ...... 24 ...... 23 ...... 11 ...... 24 ...... 24 24 23 25 25 10 09.04.09 12:57

3 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Final Biomarker report.indd 4

4 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 10. Needforfurtherstudies 9. Ethicalimplicationsofbiomarkerresearch 7. Exploitingadvancedtechnologicalplatformstostudycommonmarkersorsignatures 12. Recommendations 11. Conclusions 8. Obstaclesinbiomarkerdiscovery Ref 13. Designingfurtherbiomarkerstudiesandfundingissues of hostresponse 12.1. 7.2. 12.2. 7.4. 7.3. 7.1. 12.3. 12.4. 12.5. 12.6. 12.10. Clinicaltrialsandmarkersstudies 12.9. 12.8. 12.7.

erences...... 38

Multiplexassessments(biosignatures) Metabolomics Proteomics Transcriptomics Drug-resistance testing Culture-based biomarkerdevelopment Mycobacterial speciation Exploratory studies Pharmacokinetic andwholebloodbactericidalstudies Breath biomarkersstudies Paediatric TBandbiomarkers Markers ofprotectiveimmuneresponse Markers relevanttotreatmentmonitoring 12.10.2. Markersstudiesduringusualpatientcare 12.10.1. PhaseIIItreatmenttrials 12.10.4. BiomarkerworkwithinnationalTBprogrammeconditions 12.10.3. Opportunitiesforcollaborativework ...... 30 ...... 31 ...... 29 ...... 35 ...... 34 ...... 35 ...... 35 ...... 36 ...... 32 ...... 36 ...... 32 ...... 34 ...... 36 ...... 33 ...... 35 ...... 35 ...... 37 ...... 37 34 34 34 28 36 36 37 09.04.09 12:57 Final Biomarker report.indd 5 A References forAnnex5 Paediatric TB: diagnostics and biomarkers A Presentations at the meeting Presentations of participants List Annex 2 Meeting agenda Annex 1 Background tothemeeting Annex 3 nnex 5 nnex 4 A5.4. A5.3. A5.2. A5.1. Bibliography forAnnex4 References forAnnex4 A4.8. A4.7. A4.6. A4.5. A4.4. A4.3. A4.2. A4.1. A3.6. A3.5. A3.4. A3.3. A3.2. A3.1.

Diagnostics andclinicalbiomarkers Special clinicalsituationsinpaediatricTB Diagnosis ofpaediatricTB Introduction Designing studiesforTBandTB/HIVbiomarkers New insightsintopromisingbiomarkersinTB:prospectsforimprovedinterventions and outlook Human correlatesandbiomarkersofTB:theTBVaccine Consortium(TBVAC) experience Immune markersinTB:resultsfromtheEUFP6-fundedVACSIS studies TB/HIV: definition,needandapproachesforidentifyingspecificbiomarkers Review ofcurrentlyavailablebiomarkersandsurrogateend-pointsinTB EU perspectiveonTBresearchandbiomarkers Overview oftheglobalTBproblemandbackgroundtomeeting Background androleofTDR Meeting outline Meeting objectivesandexpectedoutcomes Need toidentifyrelapseandstratifypatients Untapped potentialofTBtherapeuticagents Link betweenHIVinfectionandanti-TBdrugresistance Link betweenTBandHIVpandemics ...... 45 ...... 127 ...... 98 ...... 47 ...... 51 ...... 123 ...... 126 ...... 52 ...... 127 ...... 52 ...... 128 ...... 50 ...... 127 ...... 128 ...... 51 ...... 50 ...... 66 ...... 50 ...... 50 ...... 113 ...... 50 ...... 59 ...... 86 ...... 73 ...... 107 130 49 09.04.09 12:57

5 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Final Biomarker report.indd 6

6 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Professor Alimuddin Zumla. Klein, DrOleOlesen,Professor Tom Ottenhoff, DrShreemantaParida, DrPNarayanan and Professor MarkDoherty (Chair), Professor Hannah Akuffo, Dr Abraham Aseffa, Professor Nigel Working group2(hostbiomarkers) Andrew Nunn,DrPhilipOnyebujoh, DrDick vanSoolingen and Dr Mahnaz Vahedi. Dr Robert Wallis (Chair),DrHannuLång,CamilleLocht, Professor DenisMitchison, Professor Working group1(pathogenbiomarkers) Mahnaz Vahedi organized themeeting. developed therecommendations.DrOleOlesen,Hannu Lång,DrPhilipOnyebujoh andDr The meetingwas chaired byProfessor Alimuddin Zumla. oftheworkinggroups The members Acknowledgements Andrew Nunn,Professor Tom Ottenhoff, DrMahnaz Vahedi andDrPhilipOnyebujoh. Doherty, Professor Nigel Klein,DrShreemantaParida, DrOleOlesen,HannuLång,Professor The meetingreport was written byProfessor Alimuddin Zumla,DrRobert Wallis, Professor Mark Authors Locht, Professor NigelKleinandDrPhilipOnyebujoh Tom Ottenhoff, Professor Hannah Akuffo, DrRobert Wallis, Professor MarkDoherty, Parida, DrShreemanta DrCamille Ridley. Standing(left to right): Dr Abraham Asseffa, DrOleOlesen,Mahnaz Vahedi, Professor Dr HannuLång, Sitting (left to right): Professor Andrew Nunn,Professor Alimuddin Zumla, Professor DenisMitchison andDrRobert

WHO/TDR 09.04.09 12:57 Final Biomarker report.indd 7 PMBC PPD nHBHA mRNA MIG MDR LAM IRIS PCR NK Mtb MPFACS LTBI LAG IP INH IL Ig IGRA IFN ICOS ICAM HLA HIV HAART GTP FP EU ESR ESAT ELISPOT ELISA EDCTP EC EBA DNA CXCR CXCL CRP CFU CFP CD40L BCG ATP AIDS Abbrevi

pur per pol nat O-4 Myc mes mul mon mul lat lip lym imm int iso int int imm int ind int hum hum hig gua Fra Eur ery ear enz enz Eur Eur ear deo che che C-r col cul CD4 baci ade acq oarabinomannan ent TB infection egrated project erleukin erferon-gamma release assay erferon ercellular adhesion molecule niazid ture filtrate protein ony forming unit ucible costimulator eactive protein hly active antiretroviral therapy ymerase chain reaction ural killer mework Programme ly secretory antigenic target ly bactericidal activity ified protein derivative ipheral blood mononuclear cell throcyte sedimentation rate opean Union opean and Developing Countries Clinical Trials Partnership opean Commission uired immunodeficiency syndrome yme-linked immunosorbent spot yme-linked immunosorbent assay mokine receptor mokine interleukin nosine triphosphate a phocyte activation gene nosine triphosphate tiparameter flow cytometry tidrug-resistant xyribonucleic acid -nitrobenzylhydroxylamine lle Calmette–Guérin obacterium tuberculosis senger RNA une reconstitution inflammatory syndrome unoglobulin an leukocyte antigen an immunodeficiency virus okine induced by interferon-gamma 0 ligand tions 09.04.09 12:57

7 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Final Biomarker report.indd 8

8 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 4FDC XDR WHO uPAR TST TNF tr Th TDR TBVAC TCR TB STREP SELDI-ToF s RT RNA RFLP

fou ext Wor uro tub tra tum T h Spe T c TB tub spe rev rib res sur sol onucleic acid ns-renal uble ell receptor erse transcriptase ensively drug-resistant triction fragment length polymorphism elper face-enhanced laser desorption/ionization time of flight r-month combinationsfixed-dose kinase plasminogen activator receptor erculin skin test erculosis Vaccine Consortium cifictargeted research project cial Programme for Research and Training in Tropical Diseases our necrosis factor ld Health Organization 09.04.09 12:57 with TB: Biomarkers should be able to assess several areas of clinical management for both adults and children vaccine candidate at an early stage of clinical experimental development. an of efficacy the predict may whichprotection, of correlatesfor biomarkers reliable efficacy, disease activity, cure and relapse. In vaccine development it is similarly important to identify reducethatcouldsizetheduration and clinicalof candidatestrialsnew drugof define treatmentand biomarkersspecific more and new identify todesirable highly be would it past, the successfullyin used been have end-points these While (relapses). post-therapyyears two subsequentthe during failstocleartheoftherapyendat (failures reinfections)or plustheproportion withrecurrent disease sputum whosesubjects of proportion the aretherapy anti-TB of studies in end-points classical The mechanism of action of the therapy under investigation – among various categories of TB patients. ofthe rates ofbacillary clearance andlater risk ofrelapse andrecurrences regardless– orof the type predictionvalidatedbiomarkersfacilitateearlyandsuitableto the of lack the isobstacle major a but the present situation calls for accelerated clinical development of new vaccines, diagnostics peopleand presentingdrugs, with TB who are infected with the human immunodeficiency virus.The urgency of continuedtheresistantincreasewellnumberasof inextensivelyas and TB, ofdrug-resistant cases Theimpact of tuberculosis has been aggravated (TB) in recent years by the appearance of multidrug- E have maximal sustained outputs, is highlighted. collaboration between researchers and funding agencies, in working together in a synchronized way biomarkerhurdleschallengesfuture.inofresearchandtothe Thereviewedimportance are in the and technological platforms are discussed: these will play a significant rolestudy and potentialin facilitatingfuture ones for biomarker TB disease activity, discovery treatment response and outcome. Advances in providesIt overview anbiomarkersof Commission Geneva,2008. Julyinunder Switzerland, 2–3 on European the and (TDR) TropicalDiseasesTraining in and Research for Programme Special the by summarizesreportThis deliberationsconsultationexpert biomarkersanconvened of on TB jointly in xe disease • end-points • anticipated • disease • treatment • treatment • c utive summ

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9 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 10 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted early on during chemotherapyor diagnosisinto at riskpatients groups TB requiringclassify differentto ability durationsThe of1991). treatment Council,Research might Medical improve Chest Service/British Kong Hong 1990; al., et (Balasubramanian therapy antibiotic of course shortened a require treatmentonly mightto individualsresponding early that evidence developingis countries. Therein negativehasimplications patientforsystemspressureadherence,puts turnhealth-care inwhichon For clinical management of TB, the length of anti-TB therapy (six months of directly observed therapy) resource-constrained settings. in addition to the questionable profitability of the anti-TB drug market, which will mainly be focused on seen in the context of only 1 in every 12 drugs that enter clinical trials eventually reaching the markets,clinical TB research, despite its importance, unattractive to the pharmaceutical industry. This has to be years after completion of treatment. The long duration of clinical trials that rely Currentlyon thisthe ultimateoutcome renderssuccess of chemotherapy is measured by the rate of relapse within the firsttwo 2008). and approximately 8.8 million people develop active TB each year, resulting in 1.6human immunodeficiency million pandemic (HIV) virus has leddeaths to a resurgence(WHO, ofTB in developing countries The organismsextensivelyand etal.,reportedbeenhas (Gandhi 2006). drug-resistant (XDR) (MDR) ofat least six months and analarming increase in the incidence of infections with multidrug-resistant urgently needed as existing ones are several decades old, current treatment regimens clinical haveare routine drugsa duration Newtrials. clinical improve duringcandidates drug anti-TBnovel mayassessmentof andmanagement treatment (TB) anti-tuberculosis to response the of evaluation Early 1. I Box 1.Biomarkers studies inclinicalstages ofM. tuberculosis (Mtb)inthehuman

exposure Mtb ntrodu host: • • • Mtb • from: of protection correlates and Markers

developing developing developing

i nfection infection, active disease, latency, reactivation andrelapse infection infection c

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T a Source: Rieder HL et al. (1989) Rieder Source: B

d protection isease TB Courtesy Courtesy of T. Ottenhoff a origin. suchthoseasfrom geneexpression microarrays. Finally, analytesofeithermay be hostpathogenor for study entry. End-points may be single measurements, or may be highly multiplexed biosignatures, willnotconfounded be byconcomitant illnesses ortherapies, andmay also serveasdiagnostic tests as a response to a stimulus, either in vivo or in vitro. Some markers are disease specific: such markers levels of an analyte in a clinical sample, whereas dynamic or functional assays Biomarkersmeasure anda surrogateprocess, end-points such may be classified in several ways(Box3). Static assays measure the field to advance. of predictors satisfactory be necessarilyultimate therapeutic not success. may The validation activity of surrogate disease end-points is oftherefore markers critically important early for that indicates experienceThis1991). al.,investigators, et Echt II 1992; myocardialinfarction after(CAST mortality increase to unexpectedly found was moracizine or encainide, flecainide, withcontractions these of tachyarrhythmia,todue suppressiondeathsudden of risk contractionsatfrequently peopleoccur in prematureventricularalthough example, For events.meaningfulclinically from dissociated be may However,2006). other research has indicated the ease with which apparently appropriate biomarkers acceleratepotentialindicatestoresearchsurrogatethe (Holodniy,ofend-points RNA HIV plasmaof measurementtreatment.duringwithExperience early changes protectionthat pathogenesis andor bevaluable as asurrogate abiomarker end-point, should measure an event that is directly involved in epidemiological,therapeutic,onpathophysiological scientificbasedother evidence. or end-point, To clinicalsubstituteacan for thatsurrogate end-point a ofbasis theform mayclinicaltheytrials In 2). (Box 2001) group,working interventiontherapeutic(Biomarkers a toresponses pharmacological or processes,pathogenic or biological normal indicatecharacteristics thatmeasurable are Biomarkers 2. contribute significantly to this effort. requirestheemergenceand XDR-TB, oftheMDR- acceleration ofcurrent research. Biomarkers may exposure can take years to afterbecomedisease development of obvious.and1% than lessThe typically urgencyispopulations, TB-endemic of highly in theeven global TB and HIV pandemics, including power. This problem is compounded in the case of TB vaccine trials, where the rate of incident cases, have required large sample sizes and long total durations to ensure adequate enrolment and statistical treatedpatients, andbecause they yearsoccurto upcantwo after completion oftherapy, suchtrials been the sum of failures plus relapses. Because relapses occur in only a small proportionreflect of howadequately a patient feels, functions or survives. In anti-TB treatment trials, end-pointsClinical historically trials have have historically used clinically important parameters as end-points (Box 1). These variables candidates, thereby accelerating new drug development through shorteningdrug anti-TBof novelclinical of validationtrials. in crucial be also would but non-adherence, to dueresistance drug therapy.ofstart biomarkersSuch wouldnotonlyimprove therapeutic strategies possiblyandreduce efficacy early on during chemotherapy or markers that stratify patients into risk groupsthe evenneed priorarises toto the find surrogate biomarkers(biological markers) that provide an indicationbe ofable treatmentto focus more attention on patients who have a high adherenceriskthusand treatment outcome.ofproviders Health-care resource-limitedin poor settingsthenmay treatment outcomes. Therefore a nd surrog I de a l c h a a r te end a c teris -points ti c s ofm a rkers 11 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 12 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Box 3. Box 2. Early-stage Patient• Pathogen• Single • • Static • • • • • • • • Disease individual isbeingprotectedagainstbecominginfected and/ordeveloping disease Measurable sign(s)inahostresponsetoaninfectious agentindicatingwhetherthe Correlates ofprotection Validated markers ofcorrelates ofprotection Surrogates ofprotection effect, progress,recovery, ordeath survival A characteristic orvariable thatreflectsthefinaloutcomeofdiseaseintermsfunction, Clinical end-point Surrogate end-point A groupofbiomarkers used together thatform highlymultiplexed biosignatures Biosignature responses toatherapeuticintervention normal biologicalprocesses,pathologicalorphysiological orpharmacological A measurablecharacteristic thatisobjectively measuredandevaluated asanindicatorof Biomarker (biologicalmarker) – –

Biomarker classification Definitions P epidemiological, therapeutic,pathophysiological orotherscientific evidence. A biomark

redicts clinicaloutcomeintermsofbenefit,orharmlack ofbenefit

vs vs

management

specific

dynamic

multiplex vs

host

er thatisintendedtosubstitute for aclinicalend-pointbasedon later-stage

analytes (functional)

nonspecific

clinical

drug

trials

vaccine

development Courtesy ofR. Wallis andS. Parida Courtesy ofR. Wallis clinical trials, to reduce the required sample size. for desirable are groupspatient homogenous Furthermore,regimens. treatment lengthy on placed treatment outcome by ensuring that only patients with increased risk for slow response or relapse are stratified into different groups, drug regimens canadjusted be accordingly, enhancing adherence and would allow patients to be stratified into different groups according to disease severity. If patients are biomarkerA forextentmeasureddiseaseofdiagnosiswould beat before initiation treatmentof and 4.1. Biomarkersofextentdisease of disease, treatment effect and treatment outcome (Box 4). Different types of markers can be distinguished in TB biomarker discovery, including markers of extent 4. Clini vaccines and adjunctive immunotherapies. drugs, new development of to challenge major a is end-points efficacy traditionalforrequired time preventionremainsmanagementanddrug-resistantlargely TB HIV-associated ofuntapped.and The immunotherapeuticstheandvaccines newinpotentialfor role The 2008). Fourie,Weyer& (Wallis, understoodinadequately is circumstancethis in resistancefor responsible factors pharmacological and biological the of interplay The therapy. intermittent during resistance drug anti-TB acquired of emergence the to predisposeparticularly to appears HIV/AIDS strains, TB resistanttransmitted to representsriskthisofsomeAlthough enhancedsusceptibility al.,2006). et (Gandhi XDR) and MDR regionssomehavecoinfectionassociatedin resistanceHIV anti-TBReports people.drug with (both toolslackto accurately assessmanageand risks optimize and long-termoutcomes duallyininfected TB patients (designated immune reconstitution inflammatory syndrome Physicians or IRIS). presently antiretroviralof goaldeleteriousgeneral therapy,haveHIV-infectedimmediate consequences canin reconstitution,inadequate.Immunea is TB and HIV treatmentofcombined for knowledge Current TB control programmes that hinders TB elimination. required acquired for resolution for of symptoms. illness Ensuring presentingadherence throughout commontherapy places a large most burden on the immunodeficiencyAnti-TB TB syndrome (AIDS). therapy presently must and be continued long past the TB, time for factor most predisposing the being common cases, HIV with million intertwined, 14.4 closely are and pandemics million two The 33 at 2008). estimated currently (WHO, respectively are TB and HIV of burdens global The c 3. oinfe T he c TB tion issues ca

a l needsfor m nd HIV p a ndemi a rkers c s: 13 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 14 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted success in predicting long-term clinical outcomes. their for critical be willpopulations two these on effects differentialdistinguishdrug tobiomarkers of ability The relapses.to rise give to thought is populationnon-replicating small However, the TB. are thought to outnumber non-replicating bacilli by several orders of magnitude in containedpatients in granulomas.with Multiplecavitary populations can coexist within individual patients. Replicating bacilli andbiosynthetic profiles. Latent infection is thought be dueto distinctto a non-replicatingpopulation that infection biomarkers results TB in multiple ofbacillary subpopulations developers with distinct facing anatomical challenge localizations particularand metabolic a is Itfashion. this in biomarkersused forrequired be will accuracypredictiveSubstantiallygreater identify those TB patients at low risk of relapse, for whom an ultrashort regimen might be appropriate. Validated biomarkers may ultimately also be used in management of individual patients, for example, to the interactions of combined therapies. roles (i.e. predicting success for bothdual drug serve therapiesmaybiomarkers and Some vaccination.preventiveto responsesprotective identify vaccines) ortreatment or may (LTBI) be useful to show cure in patients with active TB, assess the risk of reactivation or the effectiveness immunotherapiesof latentand vaccines. Such tools TB might infectionusedbe to identify orpredict rapid,a non-relapsing reinfection.researchFroma perspective, toolsareacceleratetoneeded development newdrugs,of or relapse and (cure), successtherapeutic activity,disease TB monitoringin important be will that adults HIV-uninfected and HIV-infected those are biomarkersviewpoint, clinicalneeded in a Fromseveralareas.into fall biomarkers TB childrenwith and pathogen and host for needs clinical The worldwide. importance,highrelapseas rates necessitate thedurationlong therapyof thatcurrentlyis employed Markers of eventual treatment outcome and especially of relapse after initial cure would be of particular 4.3. Biomarkersoftreatmentoutcome would be extremely useful in clinical trials and could allow shortening of trial duration. broughtcontrol.markersunderdiseaseiscontrolreachSuchduringtherapythelevels tochange as Markers for treatment effect will correlate with bactericidal and sterilizing activities of drugs and should 4.2. Biomarkersoftreatmenteffect Box 4. of • of • of • of • of • • There isaneedfor biomarkers: for • of bacterialclearance(clinicalend-point)–neededfor assessingpotentialdrug candidates

treatment cure disease infection immune

Biomarkers needsinthecontext of TB detecting

activity protection

outcome prognostically

–

needed

risk

of for

relapse

assessing

potential

vaccine yoatru tuberculosis Mycobacterium

candidates ( Mtb ) There are several approaches to studying markers: 4.4. Approachestostudyingbiomarkers Nettles et al.,Sonnenberg 2004; et al., 2001). 2000; al., et Mallory 2002; al., et (Benator studies independentseveral in relapse ofpredictive and of cavitary lung disease and the extent of lung tissue involved have been shown to be associated with extentthediseaseof arefrequently availablenotresource-limited in settings.Nevertheless, severity Additionallyfacilities chestX-rayskills al.,theradiologicaletandperform2007). to grading (Perrinof response,sinceimprovement chestonradiographs generally behindlagsoverall treatment response and inactive TB and even though associated with bacterial load areClinical markers,not such as extentsuitable of cavitation on chestas radiography,a cannotmarker distinguish betweenfor active treatment A range of host biomarkers have been studied (Table 1 and Box 5) using a variety of clinical specimens: provided into the challenges and difficultiesTB biomarker discovery is faced with. is Finally, insight biomarkers. for searches targeted as well as discussed, “fishing” are in assisting Additionally, emerging novel technologies involving genomic, proteomic and metabolomic approaches individualinvestigatedhavestudiesthethatevidenceof thestrength putativeofthethese markers. biomarkers (immune parameters) found in readily available body fluids, mainly serum, and it assesses Therefore 2007;Walzl Wallis,ettheal.,focus 2008). of this review 2007;range aisonof possible alternative al., host et (Perrin extensively reviewed been recently have limitations their These level. and individual markers an on value predictive sufficient have they do nor validated been not have Taken together, most currently used microbiological and clinical markers for anti-TB treatment response exploiting • targeting • targeting • cerebrospinal • whole • urine • sputum • breath. • an d lipidomics) to study common markers signatures or of response. common to study d lipidomics)

lood, a

nd specific specific

advanced

s fl erum/plasma liva uid,

known known

leural platforms

host pathogen/microbiological

a nd

clinical p (e.g. ericardial

transcriptomics,

and

immune ffusions

markers biomarkers

proteomics,

metabolomics,

glycomics

15 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 16 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Functional studiesofTBprotection Nonspecific biomarkersofimmuneactivation Breath biomarkers TB-specific biomarkers tuberculin skintest. natural killerTcells;sICAM-1, solubleintercellularadhesionmolecule type1;suPAR, solubleurokinaseplasminogen activatorreceptor;TNF, tumour necrosisfactor;TST, latent TBinfection;MGIT™, MycobacteriumGrowthIndicatorTube; M2SCC;month-twosputumcultureconversion; Mtb,Mycobacteriumtuberculosis;NA,notavailable;NKT, BCG, bacilleCalmette–Guérin;CFU;colony formingunit;ESAT, early secretoryantigenictarget;IFN,interferon;IGRA,interferon-gammareleaseassay; IL,interleukin;LTBI, Serial sputummicrobiology Chest radiography Candidate biomarker Highly multiplexedassays Functional studiesofanti-TBtreatment ELISPOT andQuantiFERON suPAR sICAM-1 C-reactive protein Neopterin M2SCC sTNF-R, granzymeBatdiagnosis sIL-2R Sputum IFN-γ NKT cellsatdiagnosis IGRA malate synthetase alanine dehydrogenase, Anti-ESAT-6, 38kDaprotein, Urine lipoarabinomannan Urine MtbDNA Sputum antigen85 Sputum antigen85 Sputum antigen85BRNA Early bactericidalactivity to positivity Serial MGIT™orBACTEC™time Serial sputumCFUcounts M2 SCC Baseline chestX-ray Baseline chestX-ray Baseline chestX-ray Baseline chestX-ray Metabolomics Proteomics Transcriptomics Whole bloodkilling Whole bloodkilling Whole bloodkilling IGRA IGRA Whole bloodkilling Whole bloodkilling Whole bloodkilling Whole bloodkilling Whole bloodkilling QFN TA b le 1.

Current status of ® Death Treatment response Anti-TB treatmentresponse,death Anti-TB treatmentresponse,relapse Anti-TB treatmentresponse Anti-TB treatmentresponse M2 SCC Active disease Culture platesdata Anti-TB treatmentresponse Extent ofdisease,treatmentresponse Infection, activedisease Anti-TB treatmentresponse Drug evaluation Treatment response Anti-TB treatmentresponse None relapse Anti-TB treatmentresponse,failureand Superior sterilizingactivity Recurrence Recurrence Relapse Relapse Recurrence TB disease TB disease TB diseaseandinfection Anti-TB treatmentresponse and M2SCC Correlation betweenserialCFUslope Anti-TB treatmentresponse Anti-TB treatmentresponse LTBI treatmentresponse Vitamin Deffect TNF monoclonalantibodyeffect Combined antiretroviraltherapyeffect AIDS effect BCG effect TST effect contacts (usingupperboundcut-off) Prediction ofdiseaseinuntreated Immune eradicationofMtbinfection Vaccine effect Association tb biomarkers positive treatment Study sizeand ayptet Wallis etal.,2001b Many patients Epsteinetal.,1998;Wallis etal.,1998 Many patients Many patients ayptet Mitchison,1993 Many patients ayptet Wallis etal.,2001a Many patients Many patients e ainsDaviesetal.,2006;Rustomjee2008a Few patients outcome 415Sonnenbergetal.,2001 Benatoretal.,2002 Malloryetal.,2000 24/175 74/930 46/938 83 Brahmbhattetal.,2006 18/36 13 Immanueletal.,2001 11/39 19/23 /3 Nettlesetal.,2004 4/237 8/21 5/18 2/40 1/18 5/18 7/14 ~60 101 100 105 168 192 6/6 5/6 30 44 15 20 40 18 31 40 10 36 38 20 15 22 50 10 12 AS.Parida, inpreparation NA Demiretal.,2002 Chanetal.,1995 Ribeiro-Rodriguesetal.,2002 Veenstra etal.,2006 Carraraetal.,2004 Cannasetal.,2008 Wallis etal.,2001b Wallis etal.,2001b Wallis etal.,1998;Wallis etal.,2000; Desjardinetal.,1999 Eugen-Olsenetal.,2002 Scott,Murphy&Gemidjioglu,1990 Bajaj,Rattan&Ahmad,1989 Lawnetal.,2001 Hospetal.,1997 Phillipsetal.,2007 Syhre&Chambers,2008; Azzurrietal.,2006 Reference Agranoffetal.,2006 Mistryetal.,2007 Janulionisetal.,2004 Wallis etal.,2003 Carraraetal.,2004 Eweretal.,2006 Saliuetal.,2006 Kampmannetal.,2006 Tena etal.,2003 Kampmannetal.,2004 et al.,2000 Cheonetal.,2002;HoftKampmann Cheonetal.,2002;Kampmann2000 Dieletal.,2008 Dohertyetal.,2002 Martineauetal.,2007 Eweretal.,2006 B. Hostbiomarkers A. Pathogenbiomarkers Box 5. immunoglobulin; IL,interleukin; Th, T helper; TNF, tumour necrosisfactor. CXCR, chemokine receptor;ELISA, enzyme-linked immunosorbentassay; IFN,interferon; Ig, -cell stimulationresponsestomycobacterialantigens 6. 5. 4. 3. 2. 1. 5. 4. 3. 2. 1. 7.

ers thatcorrelate with Th1 responses(LAG3V3) b. Antigen/antibody tests–ELISA for IgG,IgM, IgAtoM.tuberculosis antigens a. Hostproteins a. Macrophagemarkers andchemokines Interferon- T Type 2(Th2) cytokines(IL-4, IL4 c. Immunecytokines b. T-cell markers a. B-cellmarkers –antibody/M.tuberculosis antigenstudies Immune markers c. Volatile (breathmarkers) metabolites b. Liver enzymes(serum pyruvic glutamic transaminase,serum oxaloacetic glutamic transaminase, a. Adenosine deaminase Biochemical markersandliverfunctiontests rate sedimentation b. Erythrocyte a. C-reactive protein Inflammatory markers c. ChestX-ray (extentdisease) ofdisease,severity ofdisease,cavitary b. Skintests a. Symptoms (nausea,anorexia, weight loss,cough, chest pain,sputum, haemoptysis, Clinical markers Mycobacterial volatilemetabolitesinbreath b. Mycobacterial mRNAanditsfragments a. Mycobacterial DNAanditsfragments(cannotdistinguishlive fromdeadbacilli) Mycobacterial nucleicacids c. Antigen 85BRNA b. Antigen 85B a. Antigen 85 Mycobacterial antigens c. Dectin b. Phenolicglycolipid-1 a. Lipoarabinamannan Mycobacterial cellwallproducts d. Sputum culture: conversion after twoyears’ therapy c. Sputum culture: conversion after twomonths’ therapy (timetodetection) a. Mycobacterial countinsputum Mycobacterial load b. Apoptosis mediators Serological markers

b. Early bactericidal activity decreaseincolonyb. Earlybactericidalactivity forming units(CFU)insputum duringearlyphase

ii. Mark i. Mark Fas, Fas ligand, TNF- IL ii. i. ii. associatedwith i. associatedwith alkaline phosphatase) w of treatment iii. Inflammator M. tuberculosis –pathogen andhostbiomarkers -8, MIP-1 Type 1(Th1) cytokines(IL-2, IL-7, IL-12, IFN- asting, nightsweats)asting, ers thatcorrelate with Th2 IgE,CCL22) responses(sCD30, total γ productionstudies(ELISPOT, QuantiFERON®) α,

RANTES, IFN-gene STAT1, ribosomalproteinsCXCR4, CCR5,neopterin y cytokines(TNF- Th1 responses:LAG-3V3, splicevariant ofLAG. Th2 IgE,CCL22 responses:sCD30,total

α , TNF-R1, TNF-R2, TRAF2, caspase8,FLIP, bcl-2andbax α δ , IL-1 2, IL-4/IL-4 β ,IL-6, IL-10) γ) δ 2 ratios, TNF- α , IL-1 β , IL-6, IL-10)

17 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 18 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 2000). al.,It is 2000). etbelieved that (Sirgel measurements sites experimental of between bacterial and load coupled subjects with betweenmathematical andmodelling withinof data both load bacterial of to their implementation in clinical practice. Several studies have shown significantcurrently thevariation only accepted in surrogate measures measure of treatment response, there are significantand logicalmost challengesmeasurements theboth bacterialloadalthough are Thus, Charalambous,2003). & initial bacterial load and the bactericidal efficacythe of torelated thedirectly is treatmentmeasure this thattreatmentindicate anti-TBregimen (Davies of dynamics etthe asal., negativity Gillespie 2006; It also provides an explanation for the validity of the currently accepted surrogate of two-month culture culture negative that is observed in clinical practice and reported in sputumclinical render trials. to taken time longer the and cavitationrelationshipbetween the forexplanation an by measuredcomputedtomography scanning as iscorrelated cavitationwiththe bacterial load. ofThisprovides, extentfor thefirst time, The load. bacterial higher a shown with associated been is cavitation hasthat It cavitation. and conversion sputum delayed between association an indicated ResearchTuberculosisCouncilMedicalthe by Work hasUnitedDiseases ChestUnit,Kingdom, and 5.1. Thebasisandplausibilityofbacteriologicalbiomarkers hypoxia- against activity lack INH, as adapted such drugs, Some 1994). Wayne, 2004; al., et (Karakousis can be demonstrated by nutrientoxygen and/or deprivation, in broth culture or in artificial granulomas microenvironmentsin different anatomical compartments In (Mitchison,vitro, 1985). such adaptation arise spontaneously and be selected by chemotherapy, or they may represent an adaptation toto reflect specifica semi-dormant bacillarypopulation with reduced susceptibility to killing. Such bacteria may The second phase of treatment proceeds at a greatly reduced rate compared with the first.This appears Gosling, Heifets & Gillespie, 2003). activity or EBA) or during the firstfive days (termedextended (Gillespie EBA) & Charalambous, 2003; sputum colony forming unit counts(CFU) during the firsttwo days of therapy(termed earlyresistance. bactericidal Bactericidal activity can be measured in short-term trials by examining the rate of declinebacteriostatic of drugs have an important but secondary role in this phase, preventing the emergence of otherEthambutolphase.and this in roleimportant anhave killingreplicating –bacteria of capable – of magnitude, while preventing the emergence of resistance. Bactericidal drugs such as replicatingisoniazid in lung are cavities. (INH) The first that phase of bacilli treatment extracellular must active reduce this metabolicallypopulation by of several orders mainly consisting large, is burden bacillary total theonset, its At 1985). (Mitchison,characterizedmultiplephases istherapyby drug Anti-TB 1999). Mitchison, & Ellard (Fox, months 6–24 followingthe duringrelapse of absence the and therapy of The only unequivocally accepted end-points for TB clinical trials are bacteriological cure at the completion research to date on TB biomarkers is given in Table 1 and Box 5. or biosignature that will allow the new drugs to be tested in clinical trials expeditiously. A summary of becomedrugsavailable. challengebringsThisawith itdiscoveringof implementingand biomarkera and vaccinesdiagnostics, as such tools new ifcontrolled be only can TB ofemergency global The 5. C relapse as an end-point has focused biomarker discovery on a search for early indicators oftermed sterilization.sterilization. It is traditionally measured by the relapse rate. The long duration of trials to study bacilliTheeradication &Sramek, (Wayne 1994b). of this bacillary population in clinical trials has been a Mtb ndid ; others, such as metronidazole, are active despite inactivity against aerobically grownaerobically against inactivity despiteactive metronidazole, areas such others, ; a te mi c robiologi ca l m a rkers their total number, are the main determinant of sterilization. conversionnor relapse rate. This observation supports the hypothesis that specific drugs, rather than In the last study cited in Table 2, for example, the addition of ethambutol improved neither two-month Itis important to note that the number of drugs used in treatment does not correlate with either rate. Figure 1. value (18%) for use as a guide to treatment of individual patients. wasrelatively insensitive (identifyingonlyhalf of all relapses) andlacked adequate positive predictive independent predictor of an relapse, was with positivitya culturehazard ratio two-month of example,2.8 (Benator for Study,et al. Tuberculosis Consortium However, Trials 2002,). the the of marker 22 correlation). Lastly, limited rankdata Spearmanindicate by this0.038 =marker P may –0.764, also = be inverserelationship of (R anindicating value 4, inor individual2 quadrants in patients.lie In Study one but points data All trials.distinctindicatecontext.Symbols this inratesrelapse conversionand culture two-month on drug additional an of effectincremental the thereforeshows 1 Figuretrials. practice,however,individualclinicalculturewithincomparedconversionrelapseare and two-month In identified. relationshipis statistical no which without (29%), raterelapse high atypically an with depends heavily on aHowever, this singleP<0.01). study–0.753, arm= (6SH, i.e.(R ratesix-months’relapse and ratetreatmentconversionculture two-month with between streptomycin and isoniazid) (Table These2). data may be examined at three levels. Across studies, a significant relationship exists sputum culture status after two months of anti-TB therapy (Mitchison, 1993; Wallis & Johnson, 2006) is curenon-relapsingpredictor of greatesta experience isthereas which markerwithCurrently the 5.2. Mycobacterialload to these assessments. detection,rates.indicatorsadd Othermayviability,of quantitative assuch(mRNA) messenger RNA subsequent indicator failurerelapseof and good provide a can therapy months of two first theover Correlation of change in sputum two-month conversion rate with change in relapse rate –30 –10 –10 10 0 1 5051 520 15 10 5 0 –5 –10 P =0.038). single trial. A significantrelationshipwasidentifiedbySpearmanrank correlation( Each symbolrepresentsthedifference betweenexperimentalandcontrolregimenswithina EMB, ethambutol;PZA,pyrazinamide;R,rifampicin; T, thiacetazone. in2monthconversionrate Change EMB added PZA added PZA added PZA added PZA added RIF added RIF added R =–0.764, 19 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 20 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted b a E, ethambutol;H,isoniazid;R,rifampicin; S, streptomycin; T, thiacetazone;Z,pyrazinamide. Source: Wallis &JohnsonwithkindpermissionofNova (2006), SciencePublishers. Numeralsindicatemonthsoftreatment;subscriptnumeralsnumber ofweekly dosesiftreatmentisnotadministereddaily. Regimen Rifampicin added 6SH 2SHRZ/3 or5S 2HREZ/4HR 2SHRZ/4 or6S 6SHR 2SHRZ/4 or6TH 2SHR/4 or6TH 6SH 6SHR 6SHR 2SHRE/4 or6S 2SHRZ/4TH or4S 6SHR 6SHZ 2SHZ/5S and relapse T ab Standard shortcoursetherapy Pyrazinamide added Ethambutol added b le 2. 2 H 2 Z Relationship between month-two sputum culture conversion 2 2 2 2 H H H 2 2 2 2 Z Z E H 2 2 2 2 a Z

2 No. ofsubjects Month-two sputumculture 415 347 261 154 167 129 148 194 154 148 148 169 150 171 179 conversion Rate 88415 18.8 (%) 91229.0 112 49 5174 95 6.0 129 72 11816.0 168 81 7136.0 153 87 8136.0 143 13.0 6.0 159 88 338 75 29.0 82 2.6 112 269 49 92 9112 69 8136.0 143 88 0162.0 11.0 166 112 70 66 subjects No. of Relapse 6.7 5.0 2.0 Rate (%) Tripathy etal.,1983 1979 MedicalResearchService-British Council, Research Council,1978; Hong Kong Chest Medical Hong Kong ChestService-British Medical Research Councils, 1980 Councils, 1978; East African-British East African-British MedicalResearch Medical Research Councils,1974b Councils, 1973; East African-British East African-British MedicalResearch 1979 MedicalResearchService-British Council, Research Council,1978; Hong Kong Chest Medical Hong Kong ChestService-British Medical Research Councils, 1974b Councils, 1973; East African-British East African-British MedicalResearch Medical Research Councils, 1976 Councils, 1974a; East African-British East African-British MedicalResearch Benator etal.,2002 References fragments, andlipoarabinomannan other protein (LAM) antigens in urine. more uniformly reflect totalbody bacillary burden.Several studies have examined mycobacterial DNA a potential alternative source of diagnostic material that might be more readily collected,there is substantialand that inhomogeneity withinmight and among repeated specimens. Urine has been viewed as others,specimens.adequateIn produce to unablechildren, are aspatients, monitoring.such Some anddiagnosis TB forlimitingfactor a historically beenhas sputum processingof andcollecting The 5.4. Mycobacterialmarkersinurine have greater predictive value. isneeded to determine whether other species, RNA such asthose associated with dormancy, might relapsed could not be distinguished from others based on his early RNA response. Additional research rapidly from sputum compared with viable colony counts. One patient in that study who subsequently cleared more was it finding that al.,1999), et(Desjardin RNA examinedhasantigenstudy 85B One with INH-resistant infections, potentially limiting the application of this marker. (Walliset al., 2001b). However, induction would not be anticipated in patients not treated with INHor wasprevented byconcomitant administration ofrifampicin andbythehigher doses oftwoofrifalazil dying cells (Garbe, Hibler & Deretic, In a 1996). second trial, induction of antigen 85 by INH in sputum strains,INH-resistantrequires newprotein synthesis,releasetoduenotexisting ofis and protein by notinoccurdoes it InductiontooccursINH: antigendue of subjects85 42al.,etof(Wallis 1998). 4 durationincreasesofthisprotein in therapy duringfirst week predictedof the subsequent relapse in of Several studies have examined other microbial markers in sputum. Two studies have examined levels 5.3. Mycobacterialantigens:antigen85and85B systems (Epstein et al., Wallis 1998; et al., 1998). approachis limited but promising, as is experience using time to positivity in automated liquid culture replicating, rapidly killed bacteria that are (EBA) unrelated to treatment outcome. Experience with this treatment days of two removesfirst Omissionthe ofon effects Rustomjeeal., 2008a). et al.,2006; et measure (Davies outcomethe as 28 day tothroughdecline of rate the with 2, day beginningon In the second, sputum CFU counts are measured at weekly intervals during the first month of therapy or weekly, with time to sputum culture conversion by Kaplan-Meier analysis as the outcome measure. sputummicrobiology. In the first, the frequency of sampling is increased from once twiceto monthly Twoquantitative approaches have been suggested to improve the prognostic and statistical power of 5.2.1. Quantitativesputummicrobiologicalstudies in automated liquid culture systems may help clarify the basis of these divergent observations. Lowenstein-Jensen slants versus locally prepared medium). Additional studies using time to positivity populations, mycobacterial strains or specific laboratory methods(for example, the use of commercial thedrug effect. Further research is required to determine whether these reflect differencesof magnitude in clinicalthe and positivesubjects of proportion the to regard with emergedhave differences fluoroquinolone parameter.recent regionalstudies, this In of utility the limitalso mayfactors Other guide to treatment of individual patients. relapses)lackedallofandhalfonly(identifyingadequate positive predictive value(18%)a as foruse predictor of relapse, with a hazard ratio of 2.8 (Benator In Tuberculosis patients.et individual al., in Trials value 2002). However, of Consortium be Study alsoit may22, was statusfor culture relatively example, two-month insensitive that two-month indicate culture data Limitedpositivity was an independent Mtb antigen 85 by enzyme-linked immunosorbent assay (ELISA). In the first, the magnitude magnitude and the first, the In immunosorbent(ELISA). enzyme-linkedassay by 85 antigen 21 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 22 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted i.e. distinguish tissue sterilization from mere inhibition of bacterial growth and metabolism. diagnosing TB at site of care. However, there is no evidence yet and that sensitivity moderate these specificity. highThis markers is an area with of can much interest patientspredict and mayrelapse, ill lead to a practical, other non-invasive way from of cases TB differentiated that or principal analysis components logic fuzzy by identified were Profiles benzene. and hexane naphthalene, heptane, of 59 and out, ruled been detectedcommonlymosthealthyvolatilethecontrols. had organicwerecompounds derivativesAmong TB whom in suspects 19 patients, TB 23 confirmed of breath in and studiedvolatileorganicculturesabovecompounds biomarkers.of as(2007) Phillipsserveal. et before detectable are these culture visualusefulappearancecolonies.atoofnon-invasive leadmay diagnosticThis may and test for TB In o-phenylanisole. and nicotinate methyl p-anisate, methyl methyl phenylacetate, (2008): & Chambers Syhre identified by been have compounds specific Four in patient breath indicate an infection with the organisms. chromatography/mass spectrometry now makes it possible to investigate whether these metabolites biomarkers of cure and relapse (Phillips et al., 2007). The advent of solid phase frommicroextraction metabolites and gas manner.non-invasiveVolatilea in asthma non-responsive prognosisof the etc.)predict to used peroxide, be could hydrogen chemicals, (volatile contents breath exhaled that suggested been has It 5.5. Volatile mycobacterialmarkersinbreath outcome or to another surrogate end-point. yet examined the clearance of these antigens during treatment or established a relationship to clinical Kashino et al., Napolitano2008; et al., Singh2008; et al., Tessema2003; et al., experimentalNo 2002). studies with have animals in and patients TB some otherinreported antigensand beenhas mycobacterial Detection urineof in LAM 5.4.2. Annex 4, section A4.3). RepublicTanzaniaof Research(see Medical Zambia) of(Mbeya (UniversityProgramme)Zambiaand between ongoing are (tr)DNA theUnitedGermany United(Ludwig-Maximilians-Universität), onKingdom (UniversityCollege London), studies Programme-funded Framework 7th (EC) Commission resistancemutations, thus serving multiple roles in diagnosis and monitoring. Collaborative European sputum cannot be readily obtained, such as in children, and could potentially be copyadapted numbers to(Lok et al.,detect However, 2002). drug- the approach may be particularly useful in situations where sufficient for be not sensitivity amplification;may assay reaction(PCR) relationinsputumtoculture or conversion. method presentlyThe requires nestedpolymerasechain monthsstandardoftherapy.aftertwo Responsesevaluatedhaveyetnotbeen earlier pointsattime patientsof20None ofpositivehealthy diagnosisatcontrols et al., remained 2008). (Cannas positive small of presence fragments the of reported has study One 2006). Tomei, & (Umansky into tract shed urinary cells lower fromthe arising species DNA full-length from them distinguish to (tr)DNA trans-renal termed beenhave origin,renal of be to appear Theseand fraction soluble the in appear2004a). fragments, which al., et Su 2004b; al., et Su 2000; al,, et (Botezatu cellsmammalian of apoptosisto due fragments DNA mycobacterial small of urine in presence the described have studies Several 5.4.1. Trans-renal mycobacterialDNA Urine mycobacterialLAM Mtb IS6110 of patients inDNA withurine pulmonary of TB but 79% (34/43) not in urine Mtb raim my e xlie t dans T rpdy n cud e sd as used be could and rapidly TB diagnose to exploited be may organisms Mtb infection (Boehme et al., 2005; Choudhry & Saxena, 2002; 2002; Saxena, & Choudhry 2005; al., et (Boehme infection Mtb strains with low IS6110lowstrainswith Mtb Some tests can potentially be used in both settings. 1 and dehydrogenase (alanine enzymes malate mycobacterial synthetase) before treatment two directly correlated to with treatment antibodies failure In et (Azzurri al., 2006). of amount the mycobacterial to enzymes; antibody of levels with correlated be to shown been has disease TB of Severity 6.2. Antibodiestomycobacterialenzymesand 38 against adaptiveimmuneresponsethe component keyof cure.The long-term a bringingabout responsein immune the of importance the and pathogenesis its in both disease,immunological an also is TB 6.1. Interferon-gammareleaseassays(IGRAs) at present fall into two categories: mostpromising approaches (Table 1) is urgently needed. Theapproaches most vigorously developed the validation of and defined, poorly more much is analysedfar biomarkersso various the of utility practical the broader, but much is approaches of field potentialthemarkers, host at looking When 6. C conclusion of therapy et al.,(Wu-Hsieh these2001), results should be treated with caution. responsesinsometreated patients using mayELISAapparently maintainedbe for decadesafter the response,remainedbut elevated five inHowever,treatment failures(Carrara al.,et 2004). given that clinicaladequate an withpatients 13 all in monthsthree to baseline fromdeclinedantigens RD1 to patientsofstudyoneresponsesELISPOTIn activewith TB, al.,etnotreceive2006). did(Ewer INH who ELISPOTpositive and borderlineTST childrenwith14 of 7 innegative.declinedResponses to exposure TB declined following by 68% LTBI treatment, although only 6 of the children 38 converted sourceschoolchildrenfollowingpoint British from PBMCs byantigens RD toresponses (ELISPOT) be able to serve for monitoring response to therapy. In one study, enzyme-linked immunosorbent spot There is also evidence that these responses disappear rapidly on treatment, suggesting that they may positivity appears to occur relatively rapidly, allowing early identification of infectedcases. quantified)(Arend et al., 2007;Lalvani et al., 2001).These tests have the advantage that conversion to tuberculin skin testand correlate(TST) better with exposure to an index case (where exposure can be Multiple studies have shown that these responses are more specific for of the TB complex but not the bacille Calmette–Guérin vaccine(BCG) strain (designated RD antigens). orthree(T.SPOT) (T.SPOT®+, Quantiferon®-TB antigens Gold,in-tube) that are present inmembers infected individuals. The best characterized of these tests are the IGRAs that use a combination and of T-SPOT®. two mononuclear cellsare (PBMCs) incubated with have developed cellular immune response assays that detect IFN- Twoindustrialcompanies 2001). al., et (Pathan vivo in loadantigen the is vivo exmeasured cells T effectorpathogen-specific frequencyofdeterminantintracellularkeyother thewith ofpathogens,a I-restricted antigen-specificTh 1-type IFN-

IFN, interferon;IFN, Th, T helper. biomarkers • assessment • exposed individuals. t he assessment of bacterial products described in section 5); Mtb a is the cellular immune system, particularly human leukocyte antigen class(HLA) II- and class ndid TB

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- 23 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 24 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted perse (Wallis Likeet al., 1996). in whom they predict mortality; however, these deaths appeared to be related toTurgut HIV, et and al., notWallis 2006; activeet al., TB Levels1996). are highest in patients with concommitant HIV2001; al.,treatmentduring declineetinfection,diagnosisand (Immanuel Neopterin TB levels increased areat of these three studies investigating neopterin levels during anti-TB treatment are relatively small, with of treatment and relapse was also shown previously by Hosp et al. (1997). Although the sample sizes oftreatment completion were after associated levels elevated with relapse. disease, The of association extent between pulmonaryelevated forneopterin matched levels patients at the In end lesions. during levels neopterin in therapy decrease was greater the in patients,patients with limited TB HIV-uninfectedradiological lesions In compared limited.with patients bewith thereforeextensive may HIV/AIDS (Fahey et al., Its1990). prognostic value for TB-specific outcomes in HIV-coinfected patients macrophages upon stimulation with IFN from released and by produced is (GTP), triphosphateguanosine of product catabolic a Neopterin, 6.5.1. Neopterin summarized by others (Perrin et al., 2007; Walzl etSome ofal., these 2008). are discussed below. been treatmenthaveoutcome,biomarkersfor candidate as qualify also treatmentresponse,but or Selected immune parameters that have not only been shown to be markers for extent of disease and/ 6.5. Othernonspecificimmuneparametersaspredictorsoftreatmentoutcome Walzl etNo studies al., to 2008). date have established a relationship to clinical or other end-points. and decrease in response to anti-TB treatment (Demir et al., 2002; Lai et al., 1993; Mukaeextent,disease to et proportion diagnosis,inal., patientsat 2003; TB elevatedlevelsinare bloodstream. Its the therefore and integrins leucocyteinvolved forin cell ligandadhesion and a leukocyte rolling. is cells,A soluble endothelial form byof this expressed molecule, mainly sICAM, is isreleased into ICAM-1 6.4. Solubleintercellularadhesionmoleculetype1(sICAM-1) treatment outcome. havebeenshown tosensitivebe totreatment, there isevidenceno that they are useful inpredicting soluble interleukin relates (sIL)-2R to treatment response et (Chan al., Although 1995). these markers well as granzyme B have been associated with sputum conversion (Brahmbhatt as et 2al.,Similarly and 2006). (sTNF)-R1 factornecrosistumour soluble relapse. Serum conversioncultureorsputum to clearance (Ribeiro-Rodrigues et al., however,2002); the study was too small to establish a relationship IFN- 1.Table in found be canparameters immuneselected of list A treatmentresponse.anti-TB and/or disease of occur often during and immune responses TB with other pathogens, for they specific have been shown not to be associated are with markers extent these of many though Even laboratories. several by studiedbeen hastherapy anti-TB and TB activedifferentparametersimmuneduring of role The 6.3. Hostnonspecificimmuneparametersasbiomarkersfortreatmentresponse using identified been proteomic also techniques (see have section 7.2) et(Bahk markers al., 2004). antigen-specific Recently, studies. future in biomarkers antibodiesTheseshould therefore al., groundset2007). (Gennaroevaluatedfurther be predictive as clinicalbacteriologicaloron made be can before TB wellserumdiagnosisofincrease into reported disease et (Sartain The al.,levels 2006). of antibodies against some mycobacterial proteins have been a different study, high levels of antibodies against the 38 γ measured in sputum has been associated with response to therapy and bacterial and therapy to response with associated been has sputum in measured β -2microglobulin, neopterin is recognized as aprognostic indicator in -γ and is a biochemical marker of cellular immune responses.

k Da antigen were associated with advanced 6.7. IL-4/IL-4 Mtb addressed.available.thesefornowareAssaysbeto Measurement bindingneeds IgE to specific of and the chemokine CCL22. Their usefulness for monitoring effectsE of drug therapyimmunoglobulin(Ig) total or disease lymphocyte sCD30, relapseare responses of Th2 varianttorelatedmarkers splice LAG); solubleor geneactivation (a LAG-3V3 is responses Th1 with correlates that marker A 6.6. Hostproteins (study number: LSSP-CT-2005-012173). Guinea-Bissau clinicalintrial a innegativecurrentlypatients ViroGatestestedis being(Denmark) by studiesareneededtovalidate usefulness.its valueThesuPARof for thediagnosissputum-in TB of could therefore be a candidate marker for the prognosis of anti-TB treatment outcome, although more treatment was positively associated with poor prognosis and mortality during therapy. controlsLike was observedCRP, after eight monthsofsuPAR those of toanti-TBlevels suPAR treatment. in decrease Additionally,a TB, activethe levelwithpatients 101 of of suPARfollow-up Duringbefore 2002). al., et (Eugen-Olsen sputummycobacteria inof number the correlatedwithdirectly and TB activewith and is involved in cell adhesion and motility. The soluble form of this receptor was elevated in patients monocytes andmacrophages expressedbymainlyurokinase plasminogenactivatorreceptor isThe 6.5.3 Solubleurokinaseplasminogenactivatorreceptor(suPAR) as an early indicator of treatment response is uncertain. indicatethat stratification CRP by baselineat may promote equality among treatment groups. Its role be to shownassociated been with adverse have treatment mg/litre) outcome Murphy(Scott, (>20 & Gemidjioglu, These CRP 1990). factors ofmay levels baseline high Very treatment. of month one Rattan & BaynesAhmad, 1989; et Oneal., study 1986). in paediatric TB indicated that levels fall after Plit et al., CRP 1998). levels fall with therapy and are reported to correlate with clinical response (Bajaj, levels occur in patients with far-advanced disease (Bajaj, Rattan & Ahmad,Lee 1989; & Chang, 2003; receptors on macrophages and thereby facilitate phagocytosis. Serum CRP is increased in TB; highest proteinacute-phasean is CRP produced liverthebyknownto opsonizeand pathogens, bindtoCRP where facilities for measuring CRP are not available.settings health in used commonlystill is ESR CRP,althoughof discovery the sincedecreased has ESR is one of the oldest markers of diseases activity and inflammation known todate. Its routine use 6.5.2 Erythrocytesedimentationrate(ESR)andC-reactiveprotein(CRP) biomarker that requires further evaluation. promisingrepresent a mayneopterin Thereforeserumrelapse. forpredictor as levelsneopterinfor patientsactive TB study,maximumper 39 ofa consistencythe findingstheof strengthens case the IL-4 (Demissie Thiset wasal., in sharp2004). contrast to the increased IL-4 seen in recently exposed LTBI, long-established there disproportionatewasa stableincrease encoding IL-4 mRNA in with individuals in that revealed Gambia the and Ethiopia in studies when InterestIL-4in 2005b). al.,et Dheda 2005a; al.,et patients(Dheda TB fromcells bronchoalveolarisolatedlavagefreshly in also but cells, blood in only not seen isincrease this that severity of Subsequentdisease Scott (Seah, & workRook, 2000). from these projects has confirmed were all found to be raised in freshly isolated PMBCs from TB patients, and their levels correlated with London -VACSEL/VACSIS group in longitudinal cohort studies set up in 2000. IL-4 itself, IL-13presenceThe ofIL-4 and IL-4δ2 may turn out to be a more precise measure of Th2 activity directed at δ 2 ratio δ 2 in TB wasdiscovered TB in University2 CollegebytheEuropean Union(EU)-funded δ 2, compared2, withthatencoding Mtb itself. δ 2 in TB increased TB in 2 25 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 26 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted management of to patients with TB. opportunity valuable a translationthe is measurementof thisofbiomarker intosimple testa there thatwill assist physicians theirin commencing, trials facilitatesuccessful, if would, clinical Thisphenomenon. comprehensivelycharacterizethisinvestigate and drug anti-TB new several With overall conclusion that the ratio of IL-4/IL-4 followingThe results werepatientsobtained, theiral.,etandleading contacts2008). tothe(Wassie It therefore is generally accepted that the model validating the end-point should be designed on a on designed be should end-point thevalidating model the that acceptedgenerally therefore is challenging.It specificity and sensitivity biomarker host makes This therapy. of assessment the for biomarkersas cytokines as suchmarkers host single of use theregarding emergeconcerns Some 6.9. Useofsetsmarkers toincreasethepredictivevalue trials, together with assessment of EBA. welltheascombinedearly phase short,ineffectsII TB of drugandimmunotherapies forMDR/XDR may be particularly suited to exploring the dose–response relationship of second-line anti-TB drugs, as demonstrated between some drug and immune effects (Wallis et al.,The whole 2004). blood models treatment durations required and withoutcomes consistent (Janulionis MDR-TB, et al., Wallis for 2004; et those al., 2001a). toAntagonism superiorhas been were TB drug-sensitive for regimens that andwas superior sputum in two-month culture converters Two (Wallis et al., studies 2003). reported counts CFU sputum in decline the withcorrelated therapyanti-TB during whole activitybactericidal blood that found study One mycobacteria. intracellular on chemotherapy and immunotherapy of usedbe to examine pharmacokinetic/pharmacodynamic relationship aswell asthe combined effects drugs in the cultures mimic those in vivo at the time of phlebotomy. The approach thereforeinfected cellscan readilyto remove extracellular bacteria is eliminated. As a result, concentrations of administeredet al.,In using2003). whole blood rather than mononuclear cell culture the requirement for washing of Kampmann et2000; al., Kampmann 2004; et al.,Martineau et2006; al., 2007; Saliu et al.,Tena 2006; infection, and restored by antiretroviral therapy (Cheon et al., 2002; Hoft et al., 2002; Kampmann et al., people,enhanced by BCG vaccination or vitamin D administration, impaired by T-cell depletion or TST-negative HIV inferior in be to shown been has growth ofcontrol Immune 2000). al.,et Kampmann production by studies have substituted whole blood for mononuclear cells, and have used alternative of readouts replication (light intracellular of Recent al., inhibition1988). et vaccine (Cheng effectindicatormononuclearBCG ancultures of cell as served described, originally As cultures. vivo ex in intracellularmycobacteriakill to cells blood or blood of capacity theexamined havestudiesSeveral 6.8. Wholebloodkillingassays IL-4 IL-4, of expression of levels the study, ThusIL-4 severity of disease (Walzl et al., 2008). studyperformed inSouth Africa confirmed the inverse relationship between the IL-4/IL-4 months of treatment, the expression of IL-4 Moreover, riskattobeofdisease theinUnited(Demissie,2006b). patients Kingdom, TB in after six individualsstrongwithIFN- the • the • the • the •

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tients L TBI. γ were followed over time in Ethiopian TB TB Ethiopian in time over followed were . microti M. δ 2 ratio2 and in markers for week 8 sputum smear status. treatment when combining white blood cell count, serum granzyme B and serum sTNF-R as predictive responders were 88% and 67% respectively at diagnosis and 78% and 83% respectively at week 4 of analysis, Brahmbhatt etshowed al. that(2006) the percentages of correct discriminant predictions using Similarly further. ofassessed fast be and predicting slowto needs inrecurrence and outcome treatment type final cell particular this of role the therefore patients; 21 only of total a with groups, responder the in numbers sample small had study Thispatients. of percentage high a in made be machinediscriminantvector supportanalysis, classification a patientsof in into usedslow and fast wasresponders couldcells, correctly NK total and cells T NK CD3dim/CD56+ variables, two only of CD56 infected patients should prove very valuable. versusrecentinfection,usefulnesstheirHIV- andassays,optimization predictivein theand value of and environments (Ethiopia and Germany) is encouraging. Studies to address the questions of remote unknown; however, the fact that the two studies produced similar results in very different populations small and lack detailed sequential testing routines. Thus the kinetics and duration of arethe atdifferences greatly are elevated risk of TB (Diel et al., Doherty 2008; et al., but 2002), these studies are bacterialrelativelylongitudinalloadandtwo studies inuntreated contacts suggest that thehighest responders to exposed many studies been that suggest have that the magnitude to of expected the immune response be reflects the would magnitude of adults the of majority a where regions, TB-endemic in decision-making clinical to value add to tests these forpotential the reduce greatly or recent differentiate progressive can infection from testsdistantand latent or infection. theseInability todistinguish whichstatesthese two will to degree the being important most the therapy, of initiation to prior status infection diagnose to tests these of use the for exist uncertainties Similar These could be incorporated into planned and ongoing drug trials with relatively little extra expense. assess all of these issues are urgently required in HIV-infected and HIV-uninfected adults and children. data on the stability of positive and negative responses using overIGRAs time. Longitudinal studies to used,genuinelyor differentiate between cure andremission ofsymptoms. addition,In there arefew techniques assay the populations, between difference between the associationreflect of point and numberstointroducedorbe into clinical practice. notisItclear the if differences inregimen potency reducepatienttrialstofurthergenerated in successful permitusedevidence tobeofmarkers be to trial.validatingbyOnly markers contextthein new ofanti-TB clinicaldrug trialssufficient body willa clinical large-scale a ofcontext the evaluatedin been have date to none but TB forproposed been potentialnumberof Abiomarkers have al.,1996). Kennedyet Enarson,2004; later&(Jindani, Nunn indicatedhas thatthepotency oftheregimen altercan thepoint ofassociation eithertobe earlier or drugsusedinthecontinuation Evidence phasefrom(Jindani, previous NunnEnarson, & 2004). trials only thevalidated issurrogate months of cure twoin TB currently at available negativity & (Aber Nunn, Culture but1978), relapse? this measuring is dependent by on the predicted be cure stable Can 6.10. Researchquestionsarising anti-TBtherapy. They found that particulara lymphocyte subtype, namely CD3 parameters could be used to distinguish fast from slow responders to treatment modellingshowedofcombinationearly awiththat this type immuneof after Veenstra and the(2006) al.et start of (2006) al. et predictors.Brahmbhattindependentstrongest the were 30 day on BACTEC™ in positivity to days therapyand 14of day onsputum in concentration antigen85 patients.ofThe 43 of study a in analysis to identify combinations regressionmultipleof used early microbiological (2000) markersal. etWallis that by predictedreport Atreatment outcome.two predict relapsesto markers of occurring integratedcombinationshavestudies few a only far marker.singleSo a thanrather markers of set (NK) T cells, was more prominent in TB patients compared with controls, but on their own CD3 own their on controls,but withcomparedpatients TB prominentinmore was cells, T (NK) + NK T cells did not correlate sufficiently with treatment response.However, when a combination dim /CD56 Mtb + natural killer Tee are There . dim / 27 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 28 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted cell or organism. genetic, physiological, pathological and other aspects; lipidomics: is the systematicincluding organism),study of lipidsan inof a molecules,complex more in as well as free carbohydrates,instructure; glycan of complemententire (the glycomes of comprehensivestudy the is information.Glycomics advancedOthertechnological platformsglycomicssuchaslipidomicsand alsoprovidemay valuable understanding of the component networks overall involved an in disease gain and treatment to response. us allow will data metabolomic and transcriptomic, proteomic of integration The numerous samples. often requires and task laborious and difficult proteinsa diseaseparticularstateisassociated a with clinicaldiscovery, biological samples.The variety profile of and from a identification validation and of is a novel approach to biomarker discovery. This technique is able to provide a rapid protein expression The use of surface enhanced laser desorption/ionization time of flight(SELDI-ToF) mass spectrometry interactions, TB disease state and treatment response. biosignatures,intoputbiologicalcontextprovide bemaycouldwhich insightand into host-pathogen by data of interpretationbioinformatics skilled combinedfromthesedatanewThemethodologiesexperts. assembled beintocould highly requiring complex, are analyses these from obtained sets data Thepromise data.andnew sectionA4.7) biomarkers4,Annex These studies ongoing(see are 2008). al., et Walzl 2008; al., et (Jacobsen authorsseveral by depth reviewedin are They 6). (Box treatmentresponse andpathogenesis can disease, ininvolved networks search component identifying targeted to lead than rather “fishing” where approaches novel to led has metabolomics and transcriptomics proteomics, as such platforms technological advanced of development recent The sign pl 7. IFN- thansensitive more be to prove may interleukin(CXCL)-10 chemokineas chemokinessuchAlternatively, or cytokines other2007). al., et Hougardy 2006a; al., the by expressed antigens improvebacteriastressundermaydifferentiation with recentdistantbetweeninfectionandet (Demissie bacteria) growing actively by highly (expressed antigens RD thecombining that suggest to data some are There readout. the as cytokines additional using (ii) and Further research in two areas may help advance this work: adding(i) additional antigens as stimulation, 6.11. Furtherresearchonhostimmunemarkers ofmultiple hostmarkers this case, the(in cytokines IFN- single marker (Wassie etIndeed, al., the latter2008). manuscript suggested that assaying expression assessment of levels of expression of multiple cytokines may prove more effective than reliance on a Annex 4, section A4.7) (Jacobsen et (see Walzlal., 2008; et interaction al., 2008). host–pathogen specific by driven signals multiple the – biosignatures by addressed of identification be may that weakness a TB-specific, necessarily not are responses these that potentialdownsidethe However,response. effectorexistingistherefore therethepresumably and stimulation, antigen vivo in reflects it sinceresponse, immune ongoing the representativeofmore be fact in may anddisease, ofprogress theintoinsight usefulprovide stimulation antigen can vitro a E tf xploiting a orms to study tures ofhos a dv a t response n c c ed te o mmon m γ , IL-4, andIL-4 c hnologi γ alone (Ruhwald et al., 2007) and 2007) al., et (Ruhwald alone a δ rkers or 2) directly ex2) vivowithout in ca l needed, in which such markers can be tested and validated in larger groups and different populations. stimulatedwasblood livewith The expression of RIN3 was found to be increased in CD8+ T cells in a gene expression study in which high risk for relapse of TB. prediction for of used profilinggene-expression be also might whole-blood thatsuggestdata These mitochondrial(NOLA3). (ASNA1), ATPsynthase3 nucleolar(ATP5G1)protein and familyA/member arsenitetransporter adenosinehypotheticaltriphosphate protein(ATP)-binding KIAA2013, (SOCS3), suppressorcytokinesignalling of chromosome3 (C14orf2), reading 14 frameopen2 (TEX264), 264 testis-expressed lymphocyte(LY6G6D), geneantigen G6D complex/locusinteractor6Rab (RIN3), 3 expressed genes to stratify the patients into their respective groups. The identifieddifferentially genesnine areofcombination Ras a and onlydiscriminantrequiredanalysis arm, study perindividuals 10 or cured recurrent, active, latentusingmicroarray TB analysis. with Althoughthesample size patients ofthissmall,study wasvery withonly of blood whole from extracted RNA in genes expressed TB cases and latently infected individuals. In a similar study, Mistry et al.identified (2007) differentially intracellular trafficking lactoferrin(CD64, and could Rab33A) discriminate successfully between active discriminant Using 2). Figure analysis, 2007; it al.,was shown et that a (Jacobsen group of PCR only three (RT) genes transcriptase predominantly reversequantitative involved in microbial defence and microarrayandanalysis combinationof alatently infected individualsby37 of those with compared were patients TB 40 from PBMCs of profiles expressiongene study recent a In new.relatively is for implications their andhuman disease. conceptTheofdifferential gene genesexpression patients TB inversus healthy individuals of expression differential and polymorphisms gene on attention 30 approximately of identification and genome human the of Elucidation 7.1. Transcriptomics three platforms of above Combinations metabolites the identificationofsmall exploitsMetabolomics Metabolomics from active TB proteins thatdistinguishlatent Differentially expressed Proteomics active TB that distinguishlatentfrom Differentially expressed genes Transcriptomics Box 6.

Advanced technological platforms for biomarker discovery Mtb (Cliff et al., 2004). More studiesMoretranscriptomicsof arethereforeal.,et2004). (Cliff Biochemical analytes Proteins <1500(metabolites RNA

Da) 100 1 2400 compounds 2400

000 0

000 transcripts000 0 ee hs focused has genes 00

000 proteins000 Courtesy ofS.Parida 29 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 30 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted approach, Sartainidentified et al. four (2006) controlswith (BahkUsingasensitivity a etsimilar al.,of 60–74% 2004). and 96–97% aspecificity of healthy and patients TB from samples serum distinguish 200 to used be could filtrate culturefrom showedidentifiedthatmycobacterialtwo andrRV3874) proteomicby antigens techniques(rRV3369 identify mycobacterial as well as host proteins that could potentially be used as biomarkers. One study function and biological effects. Recently proteomic techniques have also been used in research TB to theirinsightintovaluable giveproteins canthe structureofexpression and The respective mRNAs. intodisease processes, thefocus hasturned to theproteins that are encodedby the genes andtheir As genomics and transcriptomics have to date been inadequate with regard to comprehensive insight 7.2. Proteomics a biomarkers fordiscriminationbetweeninfectionanddiseasecausedby (82% sensitivity, 86% specificity). Further studies should be conducted with the aim of identifying of aim the with conducted be should studies Further specificity). sensitivity, 86% accuracy (82% high with controls and patients TB between correctly discriminated neopterin, and CRP transthyretin, biomarkers, three using modelling machine vector Support spectrometry. mass ToF other infective and non-infective inflammatory conditions were profiled on protein TB (Agranoffchip et arrays al., In by2006). this SELDI- study 349 serum samples from 179 TB patients and 170 Proteomiccontrols fingerprinting with of patient serum recently lead to identification of host diagnostic markers for with advanced TB disease. patientsin found particularlyproteinare kDa 38 theagainst Antibodiesserologicaltests. inpatients TB non-cavitary and differentiatecavitary fromto sera ablewere that –TrxC and MPT64 (HspX), X Hans J.Mollenkopf, Andreas Ziegler&StefanH.E.Kaufmann,Figure2and5a. 85(6), 2007,613–621,MarcJacobsen,DirkRepsilber, Andrea Gutschmidt, Albert Neher, KnutFeldmann, WithkindpermissionfromSpringerScience+BusinessMedia: patients and healthy contacts 2. Figure Host biomarkers in peripheral blood mononuclear cells from TB TB from cellsmononuclear blood peripheral inbiomarkers Host a Mtb antigens – 38 kDa PstS1 protein, heat shock protein Journal ofMolecularMedicine, Mycobacterium tuberculosis Candidate , response and might be able to predict final outcome. able to provide insight into host–pathogen specific interactions as well as disease state and treatment mycobacterial be metabolites as might well biomarker as discovery.Host TB in benefit of be might reasonthereandgoodtobelieve isother conditions thatsimilar (reviewed approachesKell, in 2006) severalanalyse to used been hasmetabolomicstechnological platforms,advanced using discovery standardizationpatientspublishedofcrucial.nobecomeAlthoughwillresults biomarker exist TB for orurine sample is taken, and food, fluid or drug intake before sample collection, etc. For clinical trials, the metabolite pattern found in individuals will vary aid greatly maydepending metabolomicsbiomarkeron research profoundlytime complementand Thus transcriptomic proteomicofand techniques. However,the 2006). day when (Kell, the techniques blood other than discriminating more metabolites of analysis the makes which amplified, thereby are proteome the in changes small and proteome the of downstream is metabolome the Additionallyspecies. acrossstructures identical have often metabolitescontrastIn 2007). al., etGreef der (van sequencesacid amino differentandnucleotide animal models can be hampered because across-species genes and biomarkerproteinsinpowerfulresearch,techniques verycomparative are analysishumansandfromsample of similar function proteomicpossess transcriptomic and Although treatment. drug and disease of consequencesbiochemical biomarkersidentify order spectrometrytheto mass in of means of urine by or serum as fluids such <1500 molecules (small metabolites small of identification the exploits Metabolomics 7.3. Metabolomics sample before applied analysis. be to need techniques fractionation protein and albumin as such proteins high-abundance byobscured be can serum in cytokines asproteins suchlow-abundance of signals asphosphorylation and glycosylation, which increase the functional complexity of the proteins. Often proteomicChallengesforremaintechniques. Proteinspost-translational undergo suchmodifications cost-effective immunoassays. proteomicsignatures for treatment response andoutcome, which then could leadto development of Multivariate • Bioinformatics • M. • Effector • Cytokines • Genome • Multiplex • Box 7. Need forsophisticateddatacollection, managementandanalysis Multiplex proteinhostresponseprofilingapproaches Transcriptomic hostresponseprofilingapproaches

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D a) in body body in a) 31 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 32 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted costly clinical trials to evaluate these markers. these markers have not been sufficiently validated to persuade fundingbodies to support lengthy and for monitoring response to treatment and of treatment outcome, much remains to be done. Currently intoputbiological acontext. Despite theidentification number a biomarkersof of that show promise C bioinformaticists will be requiredskilled to Highly interpret the enormously2008). complexal., data setset beforeWalzl the results2008; are al., et (Jacobsen outcome and responsetreatmentanti-TB picture”of “bigger the understand to required investigations,is metabolomic whichproteomicand A major challenge in biomarker research will be the analysis and integration of data from transcriptomic, 8. with resistance to microarrayprofilesidentify associatedto DNA multiplexandAfrica’sused ELISAhas Western Cape SouthinfantsBCG-vaccinated incolleaguesHanekomin Willemand by study ongoing approach,an patients TB al., were2007). evaluated inthis study atthe time ofdiagnosis. Taking complementarya ofninecellsgenes bloodinthat coulddistinguish activeLTBI,TB, recurrence cureet TB and (Mistry used. Similarly, a recent report using DNA microarrays identified candidatesignaturesbiomarkers. studywaslimited,The however, involvingthebyrelative insensitivity ofexpressionthetechniques profiles study of serum by SELDI-ToF mass spectrometry. Serum amyloid fingerprinting proteomic A and on transthyretin based conditions inflammatory were andamonginfectious otherthe differentiated from be could found TB that superiorsingletoimmunebebiomarkers.assays may (2006) al. etAgranoff Severalimplyingthathighlyrecent multiplexedTB, instudies true issuggestsame the al., et 2005). (Griffiths malaria and 2005) al., etinfection(Ockenhouse HIV 2005), al., etBurczynski 2004; al., et Multiplex assessments have proven to have significant prognostic/diagnostic value incancer (Bullinger 7.4. Multiplexassessments(biosignatures) expression profiles. gene bypredicted be can vaccination by protectioninduced or patients treated reinfection)in to or populations, and to verify the suggestions by these reports that recurrent (whether TB due to relapse clinical other reproduced in be can determine findings towhethertheseneeded research isFurther Are • Can • Assays • Assays • Have • • Reflect • Box 8. O Assist bst

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care a rker dis c o very country to country. from and site to site from significantly differ obtained be can that volumes Blood blood). as (such issues may alsoimpli impact onsuch studies asspiritual values may beattached to certain samples types unidentified biomarkers. Regardless ethical permissionsof acquisition, sample culturalfor and social yet as ofvalidation and testingA allow to worded are formsconsent that important is ittechniques, review board permissions. Where samples are collectedhi for a variety of biomarker discovery laboratory Storage of samples for future analysis must be covered by suitable participant consent and institutional 9. confounding factors such as partially treated patients and drug resistance. excludestudies, to such for needed be would TB of episode first a significantly. patients with New the presence immuneof previous asTB in a further,high proportion mattersof patients, complicates which patientswould slow down TB deficiencyrecruitment may seriouslyrates in affect host coinfection biomarker expression. HIV A further complicating offactor could prevalenceinclude high The such studies. Advanced data and sample management systems must also be in place. requiredfor this purpose constitute an additional challenge on participant recruitment and retention in collectionofsputum,serum, plasma,urinemayrequired. beRNA, and PBMCs sampleThevolumes reliableforbiomarkerscurrently isunknown, type the sample ideal the cryopreservationcells.As of for supplynitrogen liquid and supply electricalreliablerequire will samples of storage andavailable facilitiesfingerprintingLaboratoryculture, be for sensitivity must testingDNA drug trial). and a such of conduction successful for required is that programme control TB functional a of establishment the(demonstrating acceptable be Follow-up shouldrates cure andfeasible cases. be must years two TB than more for pulmonary culture-positive first-time, HIV-uninfected of numbers sufficient requirement of the by dictated be instance, first the in will, sites field site of choice and preparedness.The funding substantial require would nature, in multisite or site one at whether trial, a Such at enrolment. The patients should ideally all fall under the same trial protocol to ensure standardization.as true relapse rather than reinfection. This also implies that patients would have to be culture positive DNA fingerprinting of strains is necessary forboth disease episodes to enable the correct characterizationconducted. incidence settings (Wang et al., 2007; Warren et whereal., such2002), studies would presumably be shouldnotedbe that frequent reinfections withdifferent bacterial strains have beenreported inhigh- and500 1000 patients, assuming a recurrence rate of 5% and a true relapse rate that is even lower. It after patientsinitial cure,withrecurrent studyawould have diseaseto include 20–40) (e.g. between collected.parametersimportantTobetothe needof achievedatawhichfor significant a number of points. Treatment adherence, drug susceptibility, concurrent illness and HIVmicrobiological characterization uniform clinical and timerequiresufficient statuswould at andmonths, 24represent just some subsequent18– the from and periodtherapypretreatment six-monthfrom throughout samplesthe and special field site characteristics. Ideally, biomarker studies for relapse would collectProspective suitablestudies patient for validation of such markers would require substantial funding, advanced logistics E t c l c a t ions of biom a r ker rese a r c h 33 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 34 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted primary from secondary (acquired) resistance, and in uncovering mixed infections. susceptibility on entry and throughout study participation. Such tests will be important in Similarly,differentiating resistance testing, using line probe or conventional assays, will be required to determine drug 12.2. Drug-resistancetesting incorporated into new standards of care. readily most thereforebe would methods these usingdeveloped end-points Surrogateworldwide. mmend systemsthese of roleclinical growing the ofrecognitionincreasing is theresince systems,culture It is recommended that culture-based biomarker development emphasize the use of automated liquid 12.1. Culture-basedbiomarkerdevelopment 12. the quest of improved therapy and the control of the TB pandemic. biomarker challenging, pursuitis this TB field researchthe of prioritized urgentlyarea must be aid to the United Kingdom Medical Research Council and the Bill & Melinda Gates Foundation. Although the World Health the Organization European (WHO), and Developing Countries Clinical Trials Partnership, biomarker biomarkers finding emphasison such placed the growing,in research by theis judged by interestTheshortened. accelerated anddrugs anti-TB new oftrialsminimizedclinical and be would atthe right betime)possible. Additionally drug tolerance and resistance due to suboptimal treatment validated and found are markers patientright the treatment rightfortherapeuticstrategies suitable new(thedevelopment willof accordingly once Only intensified. be to has prognosis clinical and Thesearch for surrogate markers that can provide primary measurements of treatment effectiveness 11. Con that are necessary for licensing of new drugs and diagnostic tests. in clinical testing of new drugs. The studies should be conducted to shouldthe provideclinical for andsmall laboratoryinitial standardsstudies that should increase progressively in size and duration,conventional as andoccurs new advanced technological platforms. Thedevelopment plan for these biomarkers using contactstheir and TB withchildren and adultsHIV-uninfected and HIV-infected in surrogateend-points and biomarkers candidate validate and evaluate further to needed urgently is Research 10. R N e eed for further studies c o c lusions a tions t o TDR

a nd the E C Some larger phase II trials will require stratification based on the number of drugs to which to drugs of number opportunity. the on based requirestratification an willtrials II andphase larger challengeSome a both represents patterns resistance of range potential wide The relapse.treatmentfailureor of end-points clinicalreachingsubjects of proportionhigher the to due efficiency clinicalThepatientsinof XDR-TB. or withresearchMDR-TB enhancedis patients,in such equivalentThe ofphase trials II for markers relevant totreatment monitoring maybestbeconducted 12.7. Markersrelevanttotreatmentmonitoring population groups. Oncethe assays are standardized these could bedeveloped asbiomarkers and tested in the relevant appropriateinperformed controlledpatientsbe to metabolitesneeds breath TB conditions. the of in Only preliminary studies have been performed on mycobacterial colonies. Further work to identify the 12.6. Breathbiomarkersstudies new identify candidate mayinterventions andfor interactions,subsequent study in immune/drug TB patients. of analysis the permit will volunteers healthy in Parallelstudies ofthe pharmacokinetic andwhole bloodbactericidal activityof selected interventions 12.5. Pharmacokineticsandwholebloodbactericidalstudies host immune biomarker responses, unless specificdata suggest otherwise. microbiology. It should be assumed that this brief duration of treatment will not be sufficient to detect quantitativesitesforfamiliarmethodswithatsputum TB,drug-sensitive patients withconductedin bactericidalpharmacokineticactivity,blood andwholeexploratorybe sampling.studies Thesecould (EBA), microbiology sputum quantitative using drugs, anit-TB second-line to relationship response dose– theexample, forassess, maystudies Such arm). per subjects(15 small and exposure) drug Forsome markers oftreatment efficacy, initial studies may be required that(e.g. are one-week short 12.4. Exploratorystudies diagnostic potentialconfounder. a as recognized increasingly are mycobacteria non-tuberculous with cultures mixedculture, liquid of useincreasing the with as,essential, be also will speciationMycobacterial 12.3. Mycobacterialspeciation and functional biomarkers should be included. detection in automated liquid culture systems. The study sizesto willtime andbe quantitativesufficientlyCFU both using smallperformed thatbe shouldmicrobiologybothquantitative sputum on static immunetreatment).hostandmarkers Pathogen-based evaluated. should beboth Biomarkers based 12–18afterfollow-up months one-year minimumof a (i.e.relapse regardingtreatmentand success clinical good practiceand good at laboratory practice levels. trials These trials conductmust be of sufficient to duration to collect expertisedata laboratory and clinical necessary biosafety the and appropriate containment) (including facilities laboratory populations, patient appropriate of availability the on dependent be willtrials these for selection Sitepatients. other to importance of knowledge consideredethical ifother treatment options donot exist and ifthe study islikely to increase medical be probably will trialsimmunotherapies. Suchand drugs ofcombinationslimited of or drugs of single consisting regimens with possible be will trials II phase Smaller drugs. anti-TB injectable other fluoroquinolones or the as such drugs, key toresistance of presence the on possibly resistantand Mtb is 35 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 36 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted A4.4). A4.4). section 4, Annex in described studies moxifloxacin the of course the in arisenhave thatquestions SouthAmerica,willrequired. be Studies inallthree regions particularlymaybe helpful toresolve the or Asia either and Africa conducted studies, in two appropriate. least be At also may modifications) Alternatively, markers that will be routinely evaluated in the course of usual patient care (or with minor 12.10.2 Markersstudiesduringusualpatientcare specimens will be favoured. bankedusing sub-study case–control evaluatedretrospectivelya be in canrelapse, biomarkersthat of end-point the reach will patients of number small a only Becauserelapse. true reinfectionfrom testedmustberestrictionother bymethodsor distinguish to fragment length polymorphism(RFLP) recurrence.TB sufficient adequate collect size of Initialnumbers recurrentof to and bemust strains studies Such TB. drug-sensitive with patients in conducted best be may trialstreatment III Phase 12.10.1 PhaseIIItreatmenttrials 12.10. Clinicaltrialsandmarkersstudies recommended foradultsalsoapplytopaediatric TB. that population. A briefsummaryofthestatusisgivenin Annex 5.Basicallymostofthestudies Paediatric TB remainsunderstudiedandthereareveryfewmarkersthathavebeenstudiedin 12.9. PaediatricTBandbiomarkers need. obviousthe despite served, poorly been patientshas elderlyinfants,subjects,positivechildren and regions. In particular, assessment/validation of potential biomarkers in at-risk populations such as HIV- to be addressed, are validation of potential markers identified in differentpopulations and geographical which means addressing issues of sample storage. In all cases, neglected issues, but ones that need different markers assessed, collection of multiple sequential samples should bethe strongly of encouraged, stability andkinetics the about known is little Sinceconsidered. thereforebe should studies the of course the throughout assays blood whole simple of inclusion – retrospectively performed be can assays all However, not analyses. case–control nested by met easily most isrequirement standardsregistrationhighsameasthe trials,includefunctionalstaticandbothand biomarkers: this to conducted be also should studieshowever, useful,such most be Toguaranteed. not is studies such insuccess that caveat necessary the with individuals– exposedof numbers large inanalyses relapse.lessA expensive and faster approach than phase IIIvaccine trials isto conduct retrospective responsessuccessfulpredictivewithcomparedcure,oflatency andbe can data the ifparticularly – a expected,informationtheinformend-points tosmallnumber ofable other studiesgathered be may with Even tried. be should above, noted as biomarkers, functional and static both incorporating small enough that in-depth analysis is possible, and to this end, where possible a variety of obtainedphasestudies,inIIapproaches thoughthey bestindicative.at will be studies,PhaseII however, willbe phaseIII trial, due to the difficulty of defining protection in the general population. Some data may be vaccinestudies,areunlikelywe obtainconclusivetountil completion thefirst datasuccessfulthe of For needed. be will approaches different responses, immune protective of biomarkers identify To 12.8. Markersofprotectiveimmuneresponse the following. weremade that call for an innovative change to funding important issues. These suggestions include research,crypticguaranteedproviding andfunding.marketnoduplicitySuggestionsfollow-up leads consultationcurrentcompetitivetheexpertnotedthat researchThe fundingwork. continue theand sustain tothereafter funding of lack of because forwardtaken be cannotbiomarkerspromising on limitednumber ofapplications get funded for three- aorfive-year duration. Preliminary a data obtained only funding, of availability restricted and the equipment for atmosphere scientific competitive expensive a require With reagents. that technologies state-of-the-art the involve studies Markers These factors influence the direction of future studies. infected people, and those in confined institutions have ethical considerationschildren,HIV- inStudies agencies). funding thatof agenda might priority the be andexpertise, laboratorystudies,restrictive. practice facilities, clinical trials limitations on amount of blood taken and frequency of bleeding, cohort laboratory good availability, and clinical practice scientific interest, good with appropriate sites study Currently,markerssurrogate andstudies numbervariables areofdependent end-point a(funding on s 13. this report. biomarkers studies in adults and children. These can be used for studying all the markers described in There is an urgent need for clinical trials of newer anti-TB drug regimens to be conducted in parallel with studies will be highly dependent on the outcomes of earlier phase studies. such implementationof and design The conditions.programme undertreatment patient modify to beessential. Finally, the equivalent of phase trials IV may beconsidered in which abiomarker is used serumsamplesregularat intervals forstudies oftranscriptomics, proteomics, andmetabolomics will required,yearfollow-upofwillbe makingprobablethe total studyduration threeyears. Collection of follow-up after adequatetheensure usual toprogramme provided end-point of becompletion ofsupport therapy.additional that essential A minimum be of onewill additional it case this In conditions. programme TB nationalunderbiomarkertrials III phase conduct topossible be Alternatively,may it 12.10.4 BiomarkerworkwithinnationalTBprogrammeconditions Such coordination would decrease costs and enhance study value. clinicalbiomarkersResearcha Medical Council). integrated studysitestrial the(Durban, of one with use of moxafloxacin substitution of INH(REMox Trial) for shortening the duration of chemotherapyDevelopment/European has and Developing Countries Clinical Trials Partnership-funded clinical trial on the trials drug intended plannedto test strategies or for shortening ongoinganti-TB treatment. with Currently studiesthe Global Alliance thesefor Drug TB of coordination the for arise may Opportunities 12.10.3 Opportunitiesforcollaborativework tudies To • New • g s hould be provided for a longer duration (around 10 years). roups who are willing to work together.

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37 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 38 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted funders to create a multiplier effect for coordinated funding. could be set by the EC and TDR. They could eventually partner with American, Japanese atpolitical andscientificand discussed levels Europeanby existing researchbe development and must precedence funding bodies a and issues these that thought was It contracts. the of part be of must dissemination knowledge and studies further stimulate to agencies funding with investigators of meeting terms of priorities in required of experienced, is multi-centre what partnerships of reviewcovering consultative wide expert geographical anareas. afterAnnual commissioned be should studies These Brahmbhatt Immunemarkers Setal.(2006). measuredbefore treatment predictoutcomeofintensive Botezatu Geneticanalysis ofDNAexcreted Ietal.(2000). inurine: anew approach for detectingspecific Boehme Detectionofmycobacterial Cetal.(2005). lipoarabinomannanwithanantigen-capture ELISA Biomarkers working group(2001). Biomarkers andsurrogate endpoints:preferred definitionsand Benator Rifapentine Detal.(2002). andisoniazidonceaweek versus rifampicin andisoniazidtwice Baynes Retal.(1986). response:serial The non-immune inflammatory changes inplasmairon,iron- Balasubramanian Retal.(1990). Five year resultsofa3-monthandtwo5-monthregimensfor the Bajaj G,Rattan A, Ahmad P(1989). Prognostic value of‘C’reactive proteinintuberculosis. Indian Bahk YY etal.(2004). Antigens secretedfromMycobacterium tuberculosis: identification by proteomics Azzurri tuberculosis Serological markers and of response to A et anti-tuberculosisal. (2006). of pulmonary Comparisonoftwointerferon-gammaArend SMetal.(2007). assays andtuberculin skintestfor tracing Agranoff Identificationofdiagnosticmarkers Detal.(2006). for tuberculosis by proteomicfingerprinting Aber VR, Nunn A (1978). [Shorttermchemotherapy oftuberculosis. Factors affecting relapsefollowing R eferen tudies The • phase tuberculosis therapy. Immunology,146(2):243–252. ClinicalandExperimental , 46(8Pt1):1078–1084.genomic DNAsequencesfrom cellsdyinginanorganism.ClinicalChemistry of Society Tropical MedicineandHygiene, 99(12):893–900. in unprocessedurineof Tanzanian patientswithsuspectedtuberculosis. Transactions oftheRoyal conceptual framework. ClinicalPharmacologyand Therapeutics, 69(3):89–95. randomised clinicaltrial.Lancet,360(9332):528–534. a week for tuberculosis treatmentofdrug-susceptible inHIV-negative pulmonary patients:a InvestigationLaboratory , 46(7):695–704. binding capacity, lactoferrin, ferritin andC-reactive protein.Scandinavian Journal ofClinicaland treatment ofsputum-positive tuberculosis insouthIndia.Tubercle pulmonary , 71(4):253–258. Pediatrics, 26(10):1010–1013. approach andtestfor diagnosticmarker. Proteomics, 4(11):3299–3307. Pharmacology, 19(1):199–208. treatment inapatientpopulationGuinea.InternationalJournal ofImmunopathologyand tuberculosis AmericanJournalandCriticalCareMedicine,175(6):618–627. contacts. ofRespiratory of serum. Lancet,368(9540):1012–1021. [article inFrench]. short termchemotherapy]. BulletinoftheInternationalUnion Against Tuberculosis, 53:276–280 d evelopment and training can take place.

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Resampling-based multiple testing:examples andmethods for p-value Wassie Exvivo Letal.(2008). cytokinemRNAlevels correlate withchanging clinicalstatus ofEthiopian Warren for Useofspoligotyping accurateclassificationrecurrent RMetal.(2002). tuberculosis. Wang Prediction JYetal.(2007). ofthetuberculosis reinfection proportionfromthelocalincidence. The Walzl Biomarkers Getal.(2008). for TB treatmentresponse:challenges andfuture strategies.The Wallis RS etal.(2004). TB chemotherapy: andsterilization.American betweenimmunity antagonism Wallis RS tuberculosis. etal.(2003). The duringtreatmentofpulmonary Whole bloodbactericidalactivity Wallis RS etal.(2001b). InhibitionofINH-inducedexpression ofM.tuberculosis antigen85insputum: individuals whohadrecovered fromtuberculosis. ClinicalInfectious, 33(8):1336–1340. Diseases World HealthOrganization. tuberculosis. Antimicrobial Agents andChemotherapy, 38(9):2054–2058. Clinical Microbiology&Infectious Diseases,13(11):908–914. adjustment. Wiley. overTB patientsandtheircontacts time.PLoS ONE,3(1):e1522. Journal ofClinicalMicrobiology , 40(10):3851–3853. Journal ofInfectious Diseases,196(2):281–288. Journal ofInfection, 57(2):103–109. Jul Epub2008 22. Journal andCriticalCareMedicine,169(6):771–772. ofRespiratory Journal ofInfectious Diseases,187:270–278. 45(4):1302–1304. a potentialsurrogate marker in TB chemotherapy trials.Antimicrobial Agents andChemotherapy , Annex 1. 12.10–12.30 11.40–12:10 16:15–17:30 15.45–16:15 15:00–15:45 14:30–15:00 14:00–14:30 Lunchbreak 13.00–14:00 12:30–13.00 11:10–11:40 10:50–11:10 10:20–10:50 09:40–10:00 09:20–09:40 09:00–09:20 Time Day 1 Coffee break Discussion Chairs: MarkDohertyand Alimuddin Zumla Chair: Alimuddin Zumla Coffee break Chair: Alimuddin Zumla Item TDR introductoryremarks Discussion TB biomarkersstudiesin for thefuture Immune markersintuberculosis–currentandpossibilities markers fornewdrugs,vaccinesandimmunotherapy TB/HIV. Definition, needandapproachesforidentifying Review ofcurrentlyavailablebiomarkersin TB and Discussion E Importance andbackgroundtothemeeting Welcome andintroductionsbyattendees settings. for theirvalidation/evaluation. Potentialadoptionindifferent field WGII: (pathogenbiomarkers): Existingbiomarkersandpriorities and candidatesprioritiesforresearch. WGI (hostbiomarkers):Reviewexistingandpotentialbiomarkers discussions for Thursday’s deliberations. Working groupsIandII:allocationtopreliminary biomarkers: challengesandopportunities Designing studiesfor TB and TB/HIV TB: prospectsforimprovedinterventions New insightintothepromisingbiomarkersof MEETING U perspectiveon TB researchandbiomarkers Agend E urope Closure 1stday a

Mark Doherty Robert Wallis O Philip (Robert Wallis, Chair) Working groupII (Mark Doherty, Chair) Working groupI Andrew Nunn Shreemanta Parida Tom Robert Ridley Alimuddin Zumla Name le O O lesen/Hannu Lång O ttenhoff nyebujoh 45 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 46 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 17:00–18:00 Vote ofthanks 16:45–17:00 TDRandECviewpoints 16:30–16:45 Conclusionsandconsensussummary 16:00–16:30 15:45–16:00 PresentationofWGreportsandfinalinputs 14:00–15:45 12:45–14:00 PresentationofWGdraftreportsanddiscussion 10:45–12:45 10:30–10:45 09:00–10:30 Time Day 2 Writing assignments Coffee break Final commentsanddiscussions preliminary draftreport) Working groupsIandII(discussions,prioritysettings Chair: Alimuddin Zumla Item Closure ofthemeeting Lunch break Coffee break Wallis andMarkDoherty Alimuddin Zumla,Robert Robert Ridley Philip Alimuddin Zumla Doherty Robert Wallis andMark Doherty Robert Wallis andMark Mark Doherty(WGII) Robert Wallis (WGI) Name O E veryone le O lesen/Hannu Lång O nyebujoh, Statens T.Professor Mark Doherty Italy University of Palermo Department of Biopathology Dieli Dr Francesco Nairobi, Kenya (NLTP) National Leprosy and Tuberculosis Programme Dr ChakayaJeremiah Ethiopia Addis Ababa Armauer Hansen Research Institute Dr Abraham Aseffa Sweden SAREC/SIDA Department of Research Co-operation Resource Development Division for University Support and National Hannah Akuffo Professor Temporary Advisers Annex 2. London, England St George’s, University of London Medical Microbiology, Dept. of CMM Denis A. Mitchison Professsor Lille, France Institut Pasteur de Lille Dr Locht Camille England London Institute of Child Health Great Ormond Street Hospital for Sick Children Klein Nigel Professor Copenhagen, Denmark

S erum

I nstitute (SSI) [unable to attend] L ist ofp [unable to attend] a rti c ip London, England University College London Health Centre for Infectious Diseases and International Zumla Alimuddin (Chair) Professor USA Washington, DC PPD Wallis Dr Robert Bilthoven, Netherlands Environment (RIVM) National Institute of Public Health and the National Mycobacteria Reference Laboratory Dr vanDick Soolingen Berlin, Germany Max-Planck Institute for Infection Biology K. DrParida Shreemanta Leiden, Netherlands Leiden University Medical Center Department of Infectious Diseases, C5-P TomProfessor H.M. Ottenhoff London, England MCR Clinical Trials Unit Andrew Nunn Professor Chennai, India Dr P.R. Narayanan a nts 47 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 48 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Belgium Brussels European Commission DG Research Infectious Diseases unit Dr Hannu Lång European Commissionstaff TDR Coordinator, Research for Neglected Priorities, Dr Hans Remme TB Department Coordinator, and Drug Resistance, TB/HIV Stop Dr Paul Nunn Director, Stop TB Department Dr Mario Raviglione Director, TDR G. DrRidley Robert World HealthOrganizationstaff [unable to attend] [unable to attend] [unable to attend] Belgium Brussels European Commission DG Research Infectious Diseases unit Dr Ole Olesen infected TB patients, TDR Scientist, Evidence for treatment policy for HIV- Dr Mahnaz Vahedi Stop TB Department Medical Officer,TB Strategy andOperations, Dr Onozaki Ikushi infected TB patients, TDR Leader, Evidence for treatment policy for HIV- DrOnyebujoh Philip Chukwuka WHO Initiative for Vaccine Research HIV VaccineScientist, Initiative, WHO-UNAIDS Dr Fruth Josef Ulrich [unable to attend] with TB: Biomarkers should be able to assess several areas of clinical management for both adults and children vaccine candidate at an early stage of clinical experimental development. an of efficacy the predict may whichprotection, of correlatesfor biomarkers reliable efficacy, disease activity, cure and relapse. In vaccine development it is similarly important to identify reducethatcouldsizetheduration and clinicalof candidatestrialsnew drugof define treatmentand biomarkersspecific more and new identify todesirable highly be would it past, the successfullyin used been have end-points these While (relapses). post-therapyyears two subsequentthe during failstocleartheoftherapyendat (failures reinfections)or plustheproportion withrecurrent disease sputum whosesubjects of proportion the aretherapy anti-TB of studies in end-points classical The among various categories of TB patients. andrecurrences regardless– oformechanism the type of action of the therapy under investigation – validated andbiomarkers suitablefacilitateto predictionearlytheratesthe clearanceofof bacillary laterofand relapse risk of lack the is obstacle major a but drugs, and diagnosticsvaccines, new developmentof clinical accelerated for calls situation present the of urgency are who The HIV. TB with with presenting infected people of number in increase continued the as well as TB, of cases impactThe oftuberculosis hasbeen aggravated inrecent years bythe appearance andofXDR MDR currently not possible to predict which group of patients with latent infections will reactivate. is it andunknownreactivation aredisease.Mechanismsofclinical manifestwith progress andthey which are notoriously unreliable. Latent infections are difficult to monitor and are only dealt with when active depend TB on subjective assessment of clinical signs or the patient’s perceptionof well-being, often missed by sputum microscopy. Clinical methods of monitoring the management of patients with those with extrapulmonary TB, and thosechildren, with IRIS and– have adults few HIV-positivetubercle bacilli– patients in their of sputumgroups andSeveral aremissed. are cases TB active of 40% culture.Onaverage of peopleonly with60% active are TB diagnosed by routine microscopy –nearly diagnosis still relies on the 120-year-old method of sputum examination for acid-fast mycobacteria and annually worldwide (WHO, 2008). Many people with TB go undiagnosed infectiouseach disease, majorcontributing yeartosignificant global morbidity mortality,and a killing moreand than million1.6 as people globally persists accurate TB chemotherapy, effective inexpensive, of availability the Despite Annex 3. academics, research institutions, policy-makers and funding agencies. of developmentefforts andresearch theinform will that list priorityresearch a developto and HIV agreedto hold an informal consultation jointly to EC, identify the unit,research DiseasesPoverty-relatedgaps in the area the of biomarkers and for TB/ TDR thatchallenges these of context the in is It with poor anticipated clinical outcomes, so that their treatment may be appropriately modified. identificationpost-licensing patientsearlythe clinicalthesetting, of biomarkers Inassistin also may disease • end-points • anticipated • disease • treatment • treatment •

r a elapse ctivity

o e

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c ( nd response linical

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kground to themeeting o utcomes

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a ppropriately) 49 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 50 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted accelerate new drug development through culturesputumconversion.month-two measurable areMarkersalsoearlierthattherapyduringmay treatmentCurrentlyclinicalandearliestuse.forvalidatedduration)the treatment marker of is effect different treatment patientswith groups stratificationinto allow requirementsof (different regimens novelanti-TB drug candidates by ensuring equality of groups in clinical trials. Such markers could also during early or diagnosis treatment into at groups withpatients different risks for TB adverse treatment outcome of would facilitate stratification validation allow of that markers baselineAdditionally, measured early during be treatment can would advance that the field relapse significantly. for Biomarkers drugs. anti-TB new develop to industry pharmaceutical the for outcomethisconstitutedisincentiveonclinicaldurationtreatment.rely afteratrialsthat of long The ultimateThe successofanti-TB therapy measuredis thebyrate of relapse within two thefirst years A3.4 for assessment of efficacy due to dependence on long-term clinical outcomes. drug, vaccine and adjunctive immunotherapeutic candidate development is the length of time required management of drug-resistant and HIV-associated TB remains largely untapped. A major challenge to The potential role of new anti-TB drugs, vaccines and immunotherapeutic agents in the prevention and A3.3 and decreasing the long development timeline. treatment success would be useful in drug regimen, vaccineof biomarkersandImmunological regard.immunotherapy this trials,in management reducingpatientfacilitate costsand researchaccelerate patientsgreatlywouldHIV-infected TB and TB inreliableused andbiomarkersRobust be could that understood.inadequately circumstanceis this inresistance forresponsible factors pharmacological and biological the of interplay The therapy. intermittent during resistance drug anti-TB acquired of emergence the to predisposeparticularly to appears HIV/AIDS strains, TB resistanttransmitted to Although some andof XDR-TB). this MDR- (both risk presumably represents enhanced susceptibility Reports in some regions have associated HIV infection among anti-TB patients with TB drug resistance A3.2 long-term outcomes in HIV-infected TB patients. preventive therapy. Physicians presently lack tools to accurately assess nuclearand manage the risks ofand activationtranscriptionoptimize factor via NF- HIV, of course the accelerate to appears TB addition, In patients. TB HIV-infected in (IRIS) consequences immediate deleterious have antiretroviral can therapy, of goal factor for TB, and TB the most common presenting illness for AIDS. Immune reconstitution, a general predisposingcommonmostthe being HIV intertwined,closelypandemicsarewith TB and HIV The A3.1 research and development. such a meeting that suggestedwould yield and urgent,important information particularly meeting on this existing of conveningbiomarkers, the and madepriorities aspectsfor biomarkers of number A

Need toidentifyrelapseandstratifypatients Untapped potentialroleofTBtherapeuticagents Link betweenHIVinfectionandanti-TBdrugresistance Link betweenTBandHIVpandemics κ B :rapid diagnosis of in TB the early stages of infection may allow targeting of shortening of shortening clinical trials. T biomarkers to contribute to the existing control activities for TB (Annex 4): of development use challengesthe biomarker and for specific and issues on experts respectively.bygivenwere Presentations 2, Annex and 1 Annex in listed are participants of list and agenda The A3.6 Expected outcomes Objectives A3.5 wo working groups looked at and discussed the following topics: host • pathogen • designing • new • development • review • host • TB • • dentification of current pathogen ( currentof pathogen dentification 1. 4. 3. 2. without and children with and o review in foradults for biomarkers thecurrent TB candidates 1. global • 5. 4. 3. 2. pathogen

t immunomodulation and IRIS). r f v ( I T T T T this area of work. of protection). protection (correlates disease and infection. HIV R R R D and HIV-negative adults and children with TB. TB. children with and adults HIV-negative and in different settings. potential adoption biomarker studies. immunotherapy. and vaccines

elapse and treatment failure, as well as new technological platforms); definition, need and approaches for identifying markers); reatment failure for application to clinical trials of new anti-TB drugs, vaccines, adjunctive ailure cases. accines and adjunctive immunotherapy; o discuss the requirements for validating the utility of candidates biomarkers. biomarkers. of candidates therequirements theutility o discuss for validating in theresearch prioritize and needed o reviewidentify research and biomarkers on ongoing relapse, reinfection progression, and activity disease foro evaluate measuring their utility equirements for validating the role, adoption and evaluation of biomarkers in HIV-positive evaluation of and in HIV-positive biomarkers equirements therole, for adoption validating research their for and the development of biomarker candidates priority on ecommendations for proteomics metabolomics) and (transcriptomics, platforms eview of new technological iscussion and list of potential biomarkers for further evaluation in the context of new drugs, list evaluation in of and thecontextof potential newiscussion drugs, for biomarkers further

Meeting outline Meeting objectivesandexpectedoutcomes b

iomarkers i b i nsights mmune

iomarkers T

o B f

c c b b s urrent ontrol iomarkers iomarkers; tudies

i a nto b

nd iomarkers s

tudies (

i biomarkers

b p n p f or iomarkers romising

otential 2

008

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a f ) and host biomarkers described in theliterature. described biomarkers host ) and dults

or b

iomarkers

c T

hallenges d B isease

f a

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linical r p hildren ole

rospects a

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etection

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eed a

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reatment c ure,

51 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 52 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted partners focuses on three functions, aiming to foster: Researchneglectedon priority needs is there functions, stewardship functional its decentralization, for with TDR research teams As based in levels.developing country nationalinstitutions. and institutional individual, at countries endemic disease in leadership build to training research traditional beyond moves Empowerment a biennial report on the status of tropical disease research. provide a neutral platform for partners to discuss and harmonize their activities. A major output will be comprehensive research needs assessments, priority setting, progress analysis and advocacy, and to Stewardshipconstitutesfacilitator asmajornew role aforTDR, knowledge and manager, tosupport developed countries. Programme, the World Bank and WHO – as well as representatives of governments of developing and includes TDR’s four cosponsors – the United Nations Children’s Fund, the ThisnewvisionUnited andstrategy Jointwasendorsed Coordinating the byTDR NationsBoard2007.in BoardThe Development the management of HIV-infected TB patientsinvolvesmanagementHIV-infected TB theofmarkersdetermining bio/surrogate of role the includingpatients withdiseases.additional specific objectiveco-morbid A developingin evidence for coinfection, TB/HIV and patients’all TB populationsfor withcare of delivery managementand case research line Evidence for treatment policy for HIV-infectedThe lines.research TB ninecreatingpatients operations, restructuredits aimshas to TDR objectives,optimizetheseachieve To treatment and In order fulfil its vision,TDR has adopted a three-pronged strategy: TDR still has a technical focus, it also leverages scale, policy and impact. emphasis on greater social contextualization of research, bringing it closer to control needs, and while infectious diseases of poverty in which disease endemic countries play a pivotal role. There is now an fostereffectiveresearchon toglobalaneffortisvision new its extended: been mandateTDR’s has its vision and strategy and is changing to meet new challenges. resulta As ofchanges inthe research environment sincewas created TDR in1975, hasrevised TDR Dr Robert Ridley, TDR, Geneva, Switzerland A4.1. BackgroundandroleofTDR Summary textsandslidesareprovided. Annex 4. research • research • innovation • 3. 2. 1.

r e s esearch on neglected priority needs – to enhance access to superior interventions. tewardship – to harmonize global research efforts mpowerment – to develop disease endemic country leadership in research

o f or

n f or

d a

evelopment ccess p roduct P resent

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e valuation

for diseaseforcontroladequatelyarenot that addressed other by a a nd tions

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s ettings ting pivotal role. diseaseendemicwhichcountriesa fosteringinplay strategy effective research– … global an effort developing countries to explore an innovative area of andresearch, developed both represents researchersfrom anda microcosm EC of TDR'sthe new TDR, togetherbringingconsultation, expert This Biomarker research also brings together important aspects of two other research lines: with TB. patientsHIV-infected and IRIS presentingwithpatients of care optimalimmunomodulation for and a • the • d

iagnostics). r ange

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t f argets

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uality-assured d iseases);

53 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 54 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 55 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 56 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 57 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 58 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted is no XDR-TB and levels of MDR-TB are low 2008). (WHO, Union, Japan and the Republic of Korea, whereas in Rwanda and the United Republicone case of of XDR-TB. Tanzania,There is a highthere proportion of XDR-TB among MDR-TB cases in the former Soviet RussiantheandFederation havingcountrieshighestthe burden.total 45 haveofleastAreported at IndiaChina, with cases, TB newall representing of MDR-TB, 4.8% ofmillion cases 0.5were There (WHO, 2008). people HIV-positive among million 0.2 and people HIV-negative in TB from deaths million leadingcountriesburdenareIndia, China,TB byIndonesia, Nigeria. AfricaandSouthTherewere 1.5 The cases. HIV-positive million0.7 and casessmear-positive new million 4.1were there these, Of In there2006, were 9.2 million new TB cases globally; the number of prevalent cases was 14.4Epidemiology million. Opportunities Such markers may also be able to complement current case-detection tools and strategies. needed to help achieve this. shorteningtrialswouldthereforeof result savings.significant Surrogate cost in markers urgentlyare US$ about cost currently trials Clinical The convergence of TB and HIV has created a new disease, requiring: Needs Context: needs,opportunityandepidemiology Dr Philip Onyebujoh, TDR, Geneva, Switzerland A4.2. OverviewoftheglobalTBproblemandbackgroundtomeeting The • There • The • There • Scaling • shortened • clinical • improved • improved • evidence • new • a strategies. s t holding; ools for TB control (e.g. treatment and diagnostics). nd implementation of new diagnostics, drugs and vaccines in the control of TB. trategies (e.g. adjunctive immunotherapy) should be used to enhance current treatment

e S d mergence

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59 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 60 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Issues and challenges for conducting clinical studies forcare TB/HIV studies clinical for andconducting challenges Issues The deliverables are: capacity. The specific objectives are to develop: their building furtherprogrammes, control TB national in embedded be will activitiesresearch The patients with additional diseases. co-morbid including TB, HIV-infectedand TB populationswithpatient all for care of delivery managementand anddeployment of evidence-for-policy in real time. Its overall objective is to optimize treatment, case infected TB patients within the context of available health systems capacity, through the development – has been set up to coordinate activities.TDR’s TB/HIV It focuses on the optimization of care for HIV- InTDR’s revised strategy, a research line – Evidence for treatment policy for HIV-infected patients TB TDR’s portfolio TB/HIV Is • Can • Can • Should • evidence • Is • evidence • use • evidence • simpler, • strategies • evidence • evidence • approaches? i r b relevant? and efficacy studies or best clinical practice? c infected TB 2013); (by infected TB patients failing first-line antiretroviral (bydrugs 2013); – ( – – s – – – p nterventions (e.g. adjunctive immunomodulation) or to complement current case detection egulatory criteria? by 2013);

trategies in resource-limited settings of high-burden countries in Africa. ontrol of TB and HIV-infected TB cases? Should new guidelines be developed based on safety e considered? What levels of monitoring are needed and are the current trial endpoints still opulations (gatifloxacin-containing four-month fixed-dose combinations studies); (4FDC)

t t sa g with IRIS and HIV-infected TB; r m o c here he ole of bio/surrogate markers and immunomodulation for optimal care of patients presenting

oncomitant use of anti-TB and antiretroviral drugs;

ptimal timing of highly active antiretroviral therapy (HAART): atifloxacin-containing(by4FDC 2010); o s s ore effective anti-TB chemotherapy regimen (rifabutin) for use with HAART; fety and efficacy of4FDC for HIV-positive and HIV-negativeTB cases (by 2011); f

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61 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 62 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 63 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 64 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 65 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 66 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted (STREPs), as well as a number of TB activities in the EDCTP. Whereas the STREPS are expected are STREPS the WhereasEDCTP. the in activities TB of number a as well as (STREPs), HIV/ and malaria to respectively).AIDS, allocatedThis made it possible to was support 3 IPs 51%and allocated 16 specific targeted and research projects was 25% (while activities 24% TB-related approximately or diseases, TB poverty-related in research to to contribution EC total the of Out objective of supporting late-stage clinical trials and capacity-building in sub-Saharan Africa. The (EDCTP). EDCTP is an independent throughorganization, based EC in The Hague thein the Netherlands, by with the supported ofcontribution beena foremost has capacity-building clinical and research Clinical research groups, and receives an EC contribution of 10 million–15 million during a five-year period. comprises10–20 averageIPtesting. human Theearly to candidatesphasesvaccinediscovery from and drug promising move jointly can that groups research complementary and high-level of mass researchconsortia that are organized asintegrated Eachof the IPscomprisesprojects criticala(IPs). Translational research has been supported by a total contribution of biological mechanisms. mostly exploring new concepts for drug or ofvaccine contribution development, EC an receiveor typical generatingand years, a threeinformation of have durationabout projects basic Individual innovation. of degree significant a with projects high-risk small, Approximately developmentpipeline.productentire the acrossprojects of portfolio mixed a establishthereby and discovery,areas,clinicalbothresearchthesetranslationalprojects asinsupportwell toas able was EC instruments,the fundingdifferent of advantage taking By TB. and malaria HIV/AIDS, diseases, oftotalinvested aEC the FP6, During FP6 that many projects of FP6 are still running. consortium behind a project. Research projects are normally funded for a period of years,3–5 meaningrequirement a thatat least three partnersfrom three different European countries are included inthe Researchgroups from any countryintheworld participate can intheprojects, although isnormallyit collaborativetoresearchseveral betweenresearchsupportdifferent from groups nations.on based is FP7 of part major the previousFPs,to SimilarEU. the development researchinand on spending funding,it should nevertheless be kept in mind that the onlyFP7 constitutes about of6% total public a total budget for health research of approximatelyresearchhealthoffor budgettotal a callsnumberforopenproposalsof specific research 2013,in to runs fromhas 2007 and topics. FP7 asan multi-year research programme, but with an annually updated work programme that presents a TheFP is the most important mechanism for research funding in the EU. It is administered by the EC participate in FP7. this field from the scientific community. In addition, this presentationin priorities gives forsome suggestions guidanceget on howto to andbiomarkers TB differentof statuscurrent the understand to is a putative future topic in FP7 in the infectious diseases area. From the EU researchperspective, funding.it is TB importantis one of the priority areas in the EC’s health research agenda, and This TB presentationbiomarkers gives a short overview of the EU Framework Programmes as (FPs) a mechanism for Brussels, Belgium Dr Ole Olesen and Dr Hannu Lång, Infectious Diseases, Health Directorate, European Commission, A4.3. EUperspectiveonTBresearchandbiomarkers € 87 million87 waschannelled todiscovery-oriented research. Theseprojects are mostly € 200 million to the European and Developing Countries Clinical TrialsPartnershipClinical CountriesDeveloping and European the to million 200 € 458 million into research of the three major poverty-relatedmajorthree the millionintoresearch of 458 € 6.1 billion. While this is a significant amount significant amount of a is6.1this billion.While € 1 million–3 million. These projects are projects These million. million–3 1 € 134 million to large multidisciplinary During the firsttwo years of FP7, approximately health research under FP7 are to: 0 olbrtv poet o T-eae rsac. hs poet hv be slce fr funding for selected been following calls have for proposals projects in research topics These such as: research. TB-related on projects collaborative 10 collaborative for theme major a research, istotal witha budget researchof more than health FP6, in As FP7. by replaced was FP6 2007, In FP7 work package. separatea as project,or the ofoverall planthebiomarkers, researchininclude onembeddedeither typically projects Thesefuture. near the in applicationsreal to lead tolikely are thatresults deliver expectedtoare programme,and the of core constitutethe – diseases) poverty-related forvaccines Tuberculosis), TB-VAC integrated for(an Medicines (New project for newNM4TB vaccines against – and TB) MUVAPRED (Mucosal IPs the TB, of aspects specific many of knowledge our increaseto establishment of projects in the following areas: of this call are focused around a common theme of vaccine research, and are expected to result in the deadlineThe for thenext call for proposals thirdtopicsThe(the is earlycallofDecember FP7) 2008. concrete result from our perspective most would be Thespecific call activities. topic planning examples future and suggestions. our of parts essential are actions future forrecommendations and meeting the of report extensive An areas.research important most the identify to and future prioritiesthesetfor to are: view ofFP7.point ECTherefore, meeting thefromtheobjectives of the underresearch Health forprogramme work the in topicbiomarker a of inclusion the justifying and is still at an early planning stage. This expert consultation on biomarkers could contribute to supporting Biomarkersfor could TB be a topic in subsequent calls for proposals in the Health programme, which address • increase • improve • development • • highly • • identification • translational • mucosal • a highly e pa

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67 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 68 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 69 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 70 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 71 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 72 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted esrmns o my e ihy utpee sgaue, uh s hs fo gn expression gene from those as such singlemicroarrays. Analytes signatures, may be beof either multiplexed host or pathogen may origin. highly End-points be entry. study may for or tests measurements, diagnostic as serve also may and therapies, or concomitant illnesses confounded by be not markers will Such specific. diseasemarkersare Some or functional assays measure a process, such as a response to a stimulus, either in vivo or in vitro. several ways (slide 9). Static assays measure levels of an analyte in a clinical sample, for whereasthe field dynamicto advance. Biomarkers and their corresponding surrogate end-points may of be classified predictors in therefore satisfactoryimportant criticallyis surrogateend-points be ofvalidation The necessarilysuccess.therapeuticultimate not may activity disease of markers early that indicates experienceThis1991). al.,investigators, et Echt II 1992; myocardialinfarction after(CAST mortality increase to unexpectedly found was moracizine or encainide, flecainide, withcontractions these of contractionsoccur frequently inpeople at risk ofsudden death dueto tachyarrhythmias, suppression prematureventricularalthough example, For events.meaningfulclinically from dissociated be may However,2006). other (Holodniy, research research has accelerate indicated the to ease with which apparently end-points surrogate appropriate biomarkers of potential the indicates RNA HIV ofplasma measurement with Experience 8). (slide treatment during early changes that and protection or pathogenesisininvolved directly is thatevent an measure shouldbiomarker a end-point, surrogate a as valuable be scientific evidence. otherepidemiological,To therapeutic,pathophysiological oron trials they may form the basis of a surrogate or end-point that can biologicalsubstitute normalfor apathogenic processes,clinical indicate or pharmacological responsesend-point, basedto therapeutica that intervention In characteristicsclinical 7). (slide measurable are markers) (biological Biomarkers prevention and management of drug-resistant and HIV-associated TB remains largely unexplored. andadjunctive immunotherapies. potentialThe role fornew vaccines andimmunotherapeutics inthe required for traditional efficacy end-points is a major challenge to development of new drugs, vaccines resistancethisincircumstance timeinadequatelyTheis understood Weyer(Wallis,Fourie, &2008). during intermittent therapy. The interplayresistance of the biological drug andanti-TB pharmacologicalacquired factors of responsible emergence for the to predisposeparticularly to appears HIV/AIDS strains, TB resistant transmitted to susceptibility enhanced represents risk this of some Although some in Reports XDR). and people.MDR resistance (both drug infectedanti-TB coinfection with associatedHIV closely haveregions dually in outcomes long-term optimize and risks manage and assessaccurately to tools Physicianspresentlylack IRIS). (designatedcoinfected people HIV/TB in reconstitution,generala goalofantiretroviral Immunetherapy, inadequate. have can is deleterious immediate TB consequences and HIV of treatment combined for knowledge Current elimination. TB Ensuring adherence throughout therapy places a large burden on TB control programmes that hinders Anti-TBtherapy presently must becontinued long past the time required for resolution of symptoms. factor for TB, and TB the most common presenting illness for AIDS. strategies. The twopresent pandemics bycontrolled adequatelyare been closelyhas epidemicintertwined, Neither with 4). HIV being(slide Africathe in mostprimarily common HIV/AIDS predisposing and 3) concentratedareCases(slide resource-constrained Asiaprimarily in in TB,settings:2). (slide 14.4millionrespectivelycases,andcurrently are estimated 33 TB at and HIV burdensofglobal The Introduction Dr Robert Wallis, PPD, Washington DC, USA and TB/HIV: definition,needandapproachesforidentifyingspecificbiomarkers A4.4. Reviewofcurrentlyavailablebiomarkersandsurrogateend-pointsinTB 73 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 74 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted culturessolidliquidandon medium, theeffectand thestudyFurtherdrug.ofresearch requiredis to difference between positive, of extent subjects the of proportion the to moxifloxacinregard with of studies have focused on solid culture media. Regional differences have also emerged in recent studiesindividual patients. Experience using automated liquid culture systems in this regard is limited, ofasall relapses) mostand lacked adequate positive predictive value (18%) for use as aguide to treatment of ratio of 2.8 (Benator However, et al. 2002,). the marker was relatively insensitive (identifying only half Study, for example, two-month culture positivity was an independent predictor of this relapse,marker withmay alsoa behazard of value in individual patients. In Study 22 of inversethe relationshipTuberculosis = (R P–0.764, = by0.038 SpearmanTrials rank correlation). ConsortiumLastly, limited data indicate indicatingan 4, or quadrants2 in lie one butpoints data indicateAllcontext.distincttrials. Symbols this in ratesrelapse andconversion culture two-month on drug additional an of effectincremental the shows therefore 1 Figure trials. clinical individual within compared are relapse and conversion without which statisticalno (29%), relationship isidentified. practice,In however, culturetwo-month arm i.e.six-months’ study treatment single(6SH, streptomycinwith relapsehighatypicallyrateanisoniazid) withand a on heavily depends However, this P<0.01). –0.753, = (R rate relapse and rate culturesignificant conversionthreelevels.relationshipstudies,Acrosstwo-month a between exists therapy (Mitchison, 1993; Wallis & Johnson,(slide 11, 2006) Table 2). These data may be examined greatestat is there experiencepredictora asnon-relapsingofcure sputum whichis culture statusmonthsafteranti-TBtwoof with marker the Currently globulin. gamma and CRP rate, sedimentation chest include These baseline. at radiography present Many(slide 10), baseline are TB. sputum and CFU count in riskand non-specific studied relapse inflammatory markers predict been erythrocyte weakly have parameters clinical that biomarkers on literature available the summarizes 1 Table term clinical outcomes. predictingtheirsuccessinlong- forcritical populations be willdifferentialtwothese oneffects drug biomarkersdistinguishoftoability relapses. to The rise give thoughtto non-replicatingpopulation is non-replicatingbacilli by several orders of magnitude in patients with cavitaryHowever,TB. the small Multiple populations can coexist within individual patients. Replicating bacilli are thought to outnumber Latentinfection isthought to duebe to distincta non-replicating population contained ingranulomas. bacillarysubpopulations with distinct anatomical localizations and metabolic and biosynthetic profiles. biomarkersthat TB ofdevelopers facingchallenge particular a is It fashion.this in used be tobiomarkers forrequired be will accuracypredictive greaterSubstantially identify those TB patients at low risk of relapse, for whom an ultrashort regimen might be appropriate. Validated biomarkers may ultimately also be used in management of individual patients, therapiesforandpreventive example,usefulvaccines)maybeor to show theinteractions to ofcombined therapies. responses to vaccination. Some biomarkersprotective identifymay serve or dualinfection, latentroles with(i.e. predictingpeople success ofin treatmentfor riskboth of effectiveness drug the the assessor reactivation TB, active with patients in cure non-relapsing rapid, a predict or identify might needed to accelerate development of new drugs, immunotherapiesare toolsandperspective, vaccinesresearch Such (slide tools6). a Fromareas. several into fall biomarkers TB for needs clinical The forneeds biomarkers Clinical contribute significantly to this effort. requirestheemergenceand XDR-TB, oftheMDR- acceleration ofcurrent research. Biomarkers may exposure can take yearsafter diseaseto development of becomeand 1%obvious. than less The typically urgencyis populationsof TB-endemic the highly globalin even TB and HIV epidemics, including power. This problem is compounded in the case of TB vaccine trials, where the rate of incident cases have required large sample sizes and long total durations to ensure adequate enrolment and statistical treatedpatients, andbecause they yearsoccurto upcantwo after completion oftherapy, suchtrials been the sum of failures plus relapses. Because relapses occur in only a small variablesproportion reflect of These how adequately a end-points. patient asfeels, parameters functions important or clinicallysurvives. In used anti-TB historically treatment have trials, trials end-pointsClinical Potential role for TB/HIV biomarkers Mtb infection results in multiple infectioninresults detection of mycobacterial (see DNA Annex 4, section A4.3). a consortium of African and European countries to investigate the usefulness detection of (tr)DNA for resistance mutations, thus serving multiple roles in diagnosis and monitoring. The EU FP7 has funded sputum cannot be readily obtained, such as in children, and could potentially be copyadapted numbers to(Lok et al.,detect However, 2002). drug- the forapproach sufficient may be be particularly useful not requiresin may situations presently sensitivity assay where method amplification; The PCR conversion.nested culture sputum to relation in or points time earlier at evaluated been yet not have Responses therapy. standard of months two after positiveremained fragments are thought to arise due to apoptosis of host cells. None of 20 patients positive at diagnosis (tr)DNA ofpatientsThe notbuturine inwithpulmonary ofhealthy TB controlsetal., 2008). (Cannas One study has reported the presence of small fragments of Trans-renal mycobacterial andDNA RNA because of easy non-invasive access of the specimen for studies on children. There several pathogen markers that can be measured in urine and interest in this is growing, particularly yet examined the clearance of these antigens during treatment or established a relationship to clinical Kashino et al., Napolitano2008; et al., Singh2008; et al., Tessema2003; et al., experimentalNo 2002). studies with have animals in and patients TB some otherinreported antigensand beenhas mycobacterial Detection urineof in LAM Mycobacterial LAM in urine Urine biomarkers predictive value. determinewhether other species, RNA such asthose associated with dormancy, might have greater distinguishednotbe response.from othersRNA hisearlyAdditional onbased research neededtois more rapidly from sputum than viable bacilli. One patient in that studyOne study haswho examinedsubsequently antigen 85B RNA (Desjardin etrelapsed al.,finding 1999), that85Bcould RNA was cleared INH-resistant infections, potentially limiting the application of this marker. with or INH treatedwithpatientsanticipatednotHowever, in be 2001b). notal.,inductionwould et prevented by concomitant administration of rifampicin and by the higher of two doses ofcells HiblerInseconda (Garbe, Deretic, &trial, rifalazilinduction 1996). of antigen byinsputumINH (Wallis 85 was resistant strains, requires new protein synthesis, and is not due to release of existing protein by dying 42subjects Induction (Walliset al.,of antigen 1998). occurs itdoes notdue 85 to occur INH: inINH- levelsthisofprotein sputuminduring thefirst week therapy of predicted subsequent relapseof 4 in have studies Two 12). (slide oflevelsexamined sputum in markers microbial other examined have studies Several Other markersmicrobial in sputum culture systems (Epstein et al., Wallis 1998; et al., 1998). automatedliquid in positivity to time usingexperience is aspromising, but limited is approach this replicating,onthat areunrelated rapidly killed bacteria (EBA) totreatment outcome. Experiencewith et al., Rustomjee2006; et al.,The omission2008a). of the firsttwo days of treatment removes effects of therapy beginning on day 2, with the rate of decline through day 28 as measure.the In the second, outcomesputum CFU counts measureare measured (Davies at weekly intervals during the first month monthlyorweekly, with time to sputum culture conversion analysisby Kaplan-Meier as the outcome sputum microbiology (slide 11). In the first, the frequency of sampling is increased from once totwice Twoquantitative approaches have been suggested to improve the prognostic and statistical power of Improving sputum microbiology laboratory methods (for example, the use of commercial versus locally prepared solid media). populations, clinical determine differences specific reflect mycobacterial whetherthesein strains or

Mtb antigen 85 by ELISA. In the first, the magnitude and duration of increased of duration and magnitude the first, the In ELISA. by 85 antigen Mtb infection (Boehme et al., 2005; Choudhry & Saxena, 2002; 2002; Saxena, & Choudhry 2005; al., et (Boehme infection Mtb IS6110 inDNA urine of 79% (34/43) Mtb tan wt lw IS6110 low with strains 75 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 76 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted and inflammatory conditions based on proteomic fingerprinting study of serum by SELDI-ToF mass by serum proteomic of fingerprinting study on based conditions inflammatory and to dimension new a bring biomarkers research. now Agranoff et metabolomics al. found( 2006) that and TB could be proteomics differentiated transcriptomics, from other using infectious platforms otherwise non-specific tests by measuring multiple parameters(slide The 16). advanced technological Finally, a small number of studies suggest that specificity platformNew fortechnical biomarker studies and higher predictive value could be achieved for value and optimization of the assays should prove very valuable. Studiesquestionsaddresstotheremote 2008; of versus recental.,2002). et al.,infection, Doherty predictive et (Diel TB of risk elevated greatly at are responders highest the that suggest contacts prospectivethese resultsconfirmed in trials.bemust Similarly, longitudinaltwo studiesuntreated in 2001), al., et (Wu-Hsieh therapy ofconclusion the after decades formaintained beapparently may ELISA usingtreatedpatients some responsesinHowever, thatgiven 2004). al.,et failures (Carrara months in all of 13 patients with an adequate clinical response, but remained elevated instudy offive patients treatment with active TB, ELISPOT responses to RD1 antigens declined from baseline to three children with borderline TST and positive ELISPOT who did not receive INH (Ewer 14et of Inal., 7one 2006). indeclined negative.Responsestochildrenconverted 38 the of 6 treatment,onlyalthough from PBMC by antigens RD Britishschoolchildren to responsesfollowing point exposure,source TB ELISPOT responsefollowing declined68% by ofLTBI study One these 18). (slide of characterized IGRAs the best are tests The responses. induced TB-specific examined have studies Several untreated HIV infection. In contrast, TB outcomes seem to be poorly predicted by these markers. todue related mortalitypredictivethis tobeof predicttheytomortality.valueappears whomMuch in valuable most appear These 1). (Table 13–15) (slides outcome TB prognosticators of as activation immune of markers nonspecific examinedhavestudies Several hostCandidate immune markers II trials, together with assessment of EBA. well as the combined effects of drug and immunotherapies for MDR- and XDR-TB in short, early particularlymaybephase suited toexplore therelationshipdose–response anti-TBofsecond-line drugs,as demonstrated between some drug and immune effects (Wallis et al.,The whole 2004). blood models treatment durations required and withoutcomes consistent (Janulionis MDR-TB, et al., Wallis for 2004; et those al., 2001a). toAntagonism superiorhas been were TB drug-sensitive for regimens that superior in two-month sputum culture converters (Wallis et al.,(slideTwo 20). 2003) studies reported bactericidalactivity during anti-TB therapy correlated with the decline in sputum CFU counts and was immunotherapychemotherapyintracellularandblood on whole foundthatmycobacteria. study One examinetoused pharmacokinetic/pharmacodynamic relationship combinedthewelleffectsasofas culturesthein phlebotomy.timemimicoftheatvivothosein approachthereforeThe readilycanbe cellsremoveto extracellular bacteriaeliminated.result,is aconcentrations As administeredof drugs culture,mononuclearrequirementcellthanrathertheinfected washing bloodoffor whole using By al., Kampmann2004; et al., Martineau2006; et al., 2007; Saliu et al., Tena2006; et al., by(slideantiretroviral 2003) 19). therapy et al.,(Cheon Hoftet Kampmannal.,Kampmann 2002; et et2002; al., 2000; BCG vaccination or vitamin D administration, impaired by T-cell depletion or HIV infection, and restoredal., Immune2000). control of growth has been shown to be lux-transformedinferior indicator strains,in or TST-negativetime to people,positivity in (Cheon BACTEC™) enhancedet al., by2002; Kampmann et substituted whole blood for mononuclear cells, and have used alternative readoutsstudiesRecent have(light production1988). al.,et (Cheng effectvaccineby BCG indicator ofan culturesas cell served vivo cultures. As originally described, inhibition of intracellular replication of M. microti in mononuclear Several studies have examined the capacity of blood or blood cells to kill intracellular mycobacteriaWhole blood killing assays in ex antigen with sputum bacillary burden at diagnosis; this may indicate a potential role as a biomarker. outcomeorto another surrogate However,end-point. one study has indicated correlationa of urinary Mtb HV onetd ujcs in subjects, coinfected /HIV Recommendations in treated patients or protection induced by vaccination can be predicted by gene expression profiles.reinfection)to orrelapse to due (whetherrecurrent TB that reports thesesuggestions by the verify clinicalpopulations, other to reproduced and in be determinetocan whetherneededfindings these is researchFurther communication,2008). personalHanekom, (W. vaccination BCG afterinfection microarrayprofilesidentifyto associatedDNA multiplex usedwith andresistance has ELISA to studybyWillemHanekom andcolleagues inBCG-vaccinatedinfants inSouthAfrica’s Western Cape wereevaluated thisinthetimestudydiagnosis.atof Taking complementaryaapproach, ongoing an patients17). TB (slide recurrence2007) distinguishLTBI,(Mistry,could TB thatcure, andTB, active usingmicroarrays DNA identified signatures involving expression profiles of nine genes blood in cells report recentSimilarly, atechniques used. the ofrelativeinsensitivity however,thelimited, bywas transthyretinspectrometry.and candidateamyloidbiomarkers.Astudy theSerumwere among The 3. 1. 2.

patient populations, laboratory facilities (including appropriate biosafety containment) and the other patients. Site selection for these trials will be dependent on the availability of appropriate options do not exist and if the study is likely to increase medical knowledge of importance to adjunctive immunotherapies. Such trials will probably be considered ethical if other treatment be possible with regimens consisting of single drugs or of limited combinations of drugs and drugs, such as the fluoroquinolones or other injectable anti-TB drugs. Smaller phase II trials will the number drugs to which a challenge and an opportunity. Some larger phase II trials will require stratification based on treatment failure or relapse. The wide potential range of resistance patterns represents both in such patients, due to the higher proportion of subjects reaching clinical end-points of conducted in patients with or MDR- XDR-TB. The efficiency of clinical research is enhanced T interactions, and may identify new candidate interventions for subsequent study in TB patients. activity of selected interventions in healthy volunteers will permit the analysis of immune/drug data suggest otherwise. Parallel studies of the pharmacokinetic and whole blood bactericidal treatment will not be sufficient to detect host immune biomarker responses, unless specific methods for quantitative sputum microbiology. It should be assumed that this brief duration of exploratory studies could be conducted in patients with drug-sensitive TB, at sites familiar with microbiology whole blood (EBA), bactericidal activity, and pharmacokinetic sampling. These the dose–response relationship to second-line anti-TB drugs, using quantitative sputum week drug exposure) and small (15 subjects per Sucharm). studies may assess, for example, F a potential diagnostic confounder. liquid culture, mixed cultures with non-tuberculous mycobacteria are increasingly recognized as mixed infections. Mycobacterial speciation will also be essential, as, with the increasing use of important in differentiating primary from secondary (acquired) resistance, and in uncovering determine drug susceptibility on entry and throughout study participation. Such tests will be care. Similarly, resistance testing, using line probe or conventional assays, will be required to using these methods would therefore be most readily incorporated into new standards of of the growing clinical role of these systems worldwide. Surrogate end-points developed emphasize the use of automated liquid culture systems, since there is increasing recognition new drugs and diagnostic tests. It is recommended that culture-based biomarker development should be conducted to the clinical and laboratory standards that are necessary for licensing of increase progressively in size and duration as occurs in clinical testing of new drugs. The studies development plan for these biomarkers should provide for small initial studies, which should use of the new technological platforms of transcriptomics, proteomics and metabolomics. The biomarkers in adults and children with and without HIV infection. These should now include the R or some markers of treatment efficacy, initial studies may be required that are(e.g. short one- he equivalent of phase II trials for markers relevant to treatment monitoring may be best esearch is urgently needed to further evaluate and validate candidate TB and TB/HIV Mtb is resistant and possibly on the presence of resistance to key Mtb 77 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 78 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 5. 4.

will be highly dependent on the outcomes of earlier phase studies. patient treatment under programme conditions. The design and implementation of such studies T metabolomics will be essential. samples at regular intervals for the new platform studies of transcriptomics, proteomics, and follow-up will be required, making the likely total study duration three years. Collection of serum the usual programme end-point of completion of therapy. A minimum of one additional year of case it will be essential that additional support be provided to ensure adequate follow-up after it may be possible to conduct phase III biomarker trials under programme conditions. In this TB treatment. Such coordination would decrease costs and enhance study value. Alternatively, these studies with ongoing or planned drug trials intended to test strategies for shortening anti- of the moxifloxacin studies described earlier.Opportunities may arise for the coordination of three regions may be particularly helpful to resolve the questions that have arisen in the course studies, conducted in Africa and either Asia or South America will be required. Studies in all of usual patient care with(or minor modifications) may also be appropriate. At leasttwo specimens will be favoured. Alternatively, markers that will be routinely evaluated in the course biomarkers that can be evaluated retrospectively in a case–control substudy using banked true relapse. Because only a small number of patients will reach the end-point of relapse, recurrent strains must be tested by RFLP or other methods to distinguish reinfection from studies must be of sufficient size to collect adequate numbers ofTB recurrence. Initial and Ph be sufficiently small thatboth static and functional biomarkers should be included. quantitative CFU and time to detection in automated liquid culture systems. The study sizes will Biomarkers based on quantitative sputum microbiology should be performed using both months’ treatment). Pathogen-based and host immune markers should both be evaluated. data regarding treatment success and relapse (i.e. a minimum of one-year follow-up after 12–18 necessary clinical and laboratory expertise. These trials must be of sufficient duration to collect he equivalent of phase IV trials may be considered in which a biomarker is used to modify ase III treatment trials may be best conducted in patients with drug-sensitive TB. Such 79 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 80 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 81 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 82 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 83 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 84 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 85 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 86 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 2007). The use2007). of a proxy marker is thus a very active field of research. a good predictor of vaccine(Agger efficacy Andersen,& 2001; Mittrucker et al., 2007; Wedlock et al., of IFN- γ support this. For example, as shown in Figure A4.1, we can deliberatelystudiesrecent Severalinduceresponse. large numbersthe ofof IFN- magnitude the or cells cytokine-producing of numbertotal the as important as least at is cells cytokine-producing the of specificity andnature the that is this for reason one hypothesizedthat been has It 2007). al., et Wedlock 2007; al., et Mittrucker 2001; sufficient monitoringfor immunogenicity,Andersen,& (Agger nothing or tellslittle about efficacy us monitoringforprimarilyusedfarbeen sovaccine studies hasthat (IFN- significant rolea –play clearly gives they when aeven – verycytokines limitedsinglemeasuring thatrecognition picturegeneral now ofis There the ongoing immune response.As a result, the technology Current consensus animalsalso strongly suggest that although IFN- anti-TNF- genetic defects in these pathways, and by the accidental reactivation of LTBI in humans who received IFN- that models animal demonstrated in been has development. It vaccine hindering greatly identified, protectionreliablecorrelatesthatagainstof fact the isserious most the but inflicts substantial economic losses acrossreasons(WHO, 2008).The the globefor this are manifold, morbidity,andceaseless mortalitybyhigh monitoring very treatment. itsandof virtue by Thus, currently endemic isonly disease inthe Althoughdeveloping 2005). countries,Andersen, & epidemics in(Doherty yearindustrialized a cases newcountriesmillion 8–10estimated arean only prevented and a vaccine antibiotics arewidelyavailable. Yet and remains itidentified, thegreat ofonekillers,million causing deathswas 2–3 and agent causative the which for diseases first the of one was TB Introduction Professor Mark Doherty, Statens Serum Institute, Copenhagen, Denmark A4.5. ImmunemarkersinTB:resultsfromtheEUFP6-fundedVACSIS studies Itappears that certainsubsets of IFN- high-yield cells are, in fact, multifunctional T cells producing IFN- cytometry (MPFACS) in another animal vaccination model support this multiparameter using flowhypothesis studies Recent efficacy. vaccine withassociated andbest be suggestto seemscells that the ELISPOT and ELISA (Bennekov et al.,– suggest2006) that usingthe –A4.1 Figurepresencein data ofsuggestedbefore. The beena hassubset it ofidea: radicalparticularlythe a respondingnot is This These data suggest that we need to look at the ofsource(s) the immune response(s) being measured. signs of early TB show the opposite pattern (Figure 2C A4.2, and 2D). increasedIFN- by much withalteracute TB their cytokine production over thecourseofinfection tocharacterizedone notso analysis.Studies carried out by the VACSIS consortium using quantitative RT for MPFACS of use the onrelied studiesthese of Both 2007). al.,et (Mittruckerresponseimmune -producing T cells that offer no detectable protection – or alternatively, we can induce a similar number γ and TNF- γ -producing T cells – and get more than reduction 90% in α treatment as therapy for rheumatoid arthritis (Winthrop, However,2006). vaccine studies in α are essential for protection and this is supported by the susceptibility of humans with γ but by an improved ratio of IFN- ofimproved ratioan by but γ -producing CD8+ cells -producing may CD8+ also beimportant for protectivea γ is essential, production of this cytokine alone is not γ to IL-4, while TB contacts who developwho contacts TB whileIL-4, to γ , TNF- Mtb numbers. α and IL-2 (Darrah et al., 2007). γ ELISA and ELISPOT) while ELISPOT) and ELISA -PCR show -PCR that patients Mtb have not yet been yetnot have Mtb that the response to specific antigens from antigens specific to response the that shownhave others) (and we 2B) and 2A A4.2, different(Figurebetween alterstimecytokinesover balance the as cells'specificity.antigen Just theincludes also phenotype Theresponding cells.the of phenotypes the about anything us tell experiments these do nor responses, antigen-specific of examinationto suited well not is simultaneously,it cytokines multiple at look can RT-PCR while – and the benefit of measuring them in context. It also highlights the limitationsTheseresults ofagain emphasize thethe importance ofexamining currentindividual cytokine markerstechnology with caution Hougardy et al., 2007). control the infection (and develop LTBI) and those who develop acute disease (Demissie et al., 2006a; protection against Alteration ofepitoperecognitionpatternin Ag85B andESAT6 hasprofoundinfluenceonvaccine-induced a units orSF Although similarnumbersofantigenreactiveinterferon(IFN)- 3355. protection. phenotype) thatproduceveryhighlevelsofIFN- shown ontherighthaveagreaterpercentageofCD4+cellsandclearlyvisiblesubpopulation(ofunknown Source: Thomas Bennekov, IdaRosenkrands, Anette Stryhn, T. MarkDohertyandPeter Andersen. Protection log Protection SFU per SFU 10per plates ELSPOT F with orwithoutaneffective adjuvant ig u U re ), thoseontheleftareprimarilyCD8+andproduceonlysmallamountsofIFN- A4.1 6 Mycobacterium tuberculosis 10 . ELISPOT results after adenovirus vaccination effective adjuvant effective Without Without 512 0.0 . EuropeanJournalofImmunology, 2006Dec;36(12):3346– Mtb γ. Itisthepresenceofthesecellsthatcorrelatesbestwith typically differs between infected individuals who individualsinfectedbetween differs typically effective adjuvant effective γ -producingcellsareinduced(spot-forming With 498 0.8 a γ percell. Those 87 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 88 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted where an immune response may exist prior to vaccination et (McShane – al.,is obvious. 2004) of this approach to vaccination studies – especially with antigens such as antigen or85A antigen 85B, simultaneously.themproducethatutility cells identifyingtheThe measuring andmultiplecytokines tothe overall immune response by stimulating cells from patient samples with different antigens and subsets their contribution antigen-specific shouldand ofT-cell phenotypeidentifythe to begin to analysis usenable MPFACS technology. current our of limits the against up coming are we here for Currently, almost nothing is known about this process, neither the kinetics nor the cell types involved, a to interleukin(IL)-4orIL-4 C showrawcytokinemRNA expression,BandDshowtheratioofmessageforinterferon(IFN)- contacts, whowerehealthyatentry, butsubsequently developed TB or TB-like symptoms. A and unstimulated peripheralbloodmononuclearcellsof TB patients(A,B)andtheirhousehold Quantitative PCRofmessengerRNA (mRNA)atentrytothestudyandnine monthslater, from journal.pone.0001522. status of Source:Wassie L etal.(2008)ExvivocytokinemRNA levelscorrelate withchangingclinical F contacts developing TB-like symptoms ig u re E thiopian TB patientsandtheir contactsovertime.PLoSONE,3(1):e1522.doi:10.1371/ A4.2 . Cytokine mRNAexpression in TB patientsandin δ2 toIL-4. a γ

possibility of better identifying correlates of immunity. theaddition in offer andhumans vaccination responseinto effectivelythe moreassess to usallow clinical definition of study cohorts careful and therobust multivariate is rest statistical must technologies analyses. these Combined, which these on requirement should basic the however, so analysis, theof quality theanalyse andto samplesusedtheyare the as technologiesgoodThese as onlyare these questions, each with specific advantages/limitations: oftheir cytokine production to other cytokines. There are multiple technologies available for resolving bestunderstoodbe thecontextininvolved, ofthecell types theirantigen the and specificity relation cytokineprofiles correlate that protectionwith better IFN- than idsuentify possible to is it that suggest data technology,the the oflimitations the despitetotal, In multiplex • MPFACS, • quantitative • r p g clinical studies where sample volume is limited; eadily and less expensively than MPFACS. roduce the markers of interest; enerate a great deal of information from a very limited number of cells – always an issue in

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89 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 90 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 91 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 92 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 93 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 94 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 95 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 96 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 97 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 98 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted at relevant sites, TB-endemic in both Africa and Asia. Foundation, etc. Future biomarker discovery and validation will needVaccine TB toGlobalPartnership, Aeras TB includeStop Foundation,GatestheMelinda & studies BillWHO, ofthose in larger cohorts and other relevant models). Activities need to be integrated with other modelsinternationalgranuloma responses,cell (macrophage/dendritic activities,responsesimmuneinnatesubsets, including T-cell Markers of Markers protection Track 1 Overview of some TBVAC1 Work Package 4 biomarkers and assays, and future plans Track 2 Track 1 Activities thus far have been organized by the Group leadership into two parallel tracks. Organization innate and adaptive immune response to We think it is important to focus on assays that measure the functional capacity of human cells in the biomarkers to fill the pipeline. new of discovery by complemented validation, and development assay into forward efforts these from assays and findings promisingmost the move to proposed has Group The validation.further need which markers, promising of set prioritized a and identified has BiomarkerGroupTBVAC The Professor Tom Ottenhoff, Leiden University Medical Centre, the Netherlands (TBVAC) experienceandoutlook A4.6. HumancorrelatesandbiomarkersofTB:theTBVaccine Consortium been useful in prioritizing and selecting or discontinuing potential markers for further evaluation. BCG vaccinees and 20 healthy controls. Structuring TB biomarker activities along these two tracks has Track 2 candidates could move to track 1 once validated in small cohorts of at least 20 TB patients, 20 High • • • High • • MIG • Granulysin • Development • Optimization, • • Gene • Discovery • Translation • Whole • Mono patients). e O vaccination. xpression (assay development in progress). -4-nitrobenzylhydroxylamine (nHBHA)-induced IFN- -4-nitrobenzylhydroxylamine (nHBHA)-induced

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CFP, culturefiltrateprotein. Focused • ELISPOT • Reduced • HBHA/ESAT-6-CFP-10-specific Chemokine • IL-4-producing • Local • • Six-day • New • PPD-induced • Multifunctional • killing assays – – downstream responses in TB (rather than assessing only IFN- V Mtb v (manuscripts submitted). 72F and HYB1 trials in Lausanne, Switzerland, and Leiden, the Netherlands, respectively ersions of these protocols were successfully used in immunolgical monitoring of the

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99 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 100 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Important goalsforfutureTBbiomarkerresearch(discovery, translationalandclinical) Immune assays Markers ofdisease 5. 4. 3. • DC-SIGN • enes f List • 2. o identify novel biomarkers or biomarker signatures that are associated with protection against 1. • Regulatory • • Large • TCR • Ratio • CD64

observational (longitudinal) studies. tailor-made biomarker sets can be developed for different vaccine- and drug-interventional or vaccine type, antigen expression and trial setting. Decision-making matrices for prioritization of T T preferably be used in settings. TB-endemic T cohorts (TB patients,cohorts (TB BCG vaccinees, LTBI and healthy controls). T Mtb staining). s susceptibility. TB disease, markers and/or associated with disease(early) activity and lack of protection/ T Mtb c p o advise on the selection of tailor-made sets of biomarkers for clinical trial monitoring based on o harmonize, standardize and apply assays for clinical trial monitoring. o adapt current biomarkers to assays that are simple, robust and reproducible and can o prioritize and validate current biomarkers as well as new markers from point 1 above in small uppress via HBHA CCL4; induces regulatory CD4+ T cells). ells in TB. robe amplification).

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101 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 102 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 103 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 104 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 105 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 106 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 2 integrated manner, which is typically not possible with classical reductionist models. basisto narrow downour focus onrelevant candidates andprocesses connecting eachanother inan explorativean on approachthis use as to usallowed have decadeTechnological thisadvancements in such platforms “-omics” determiningin different relevant genes,proteins moleculesinvolvedor pathophysiologythe disease.in the of in resulted have transcriptomics,approachesglobalproteomics, usemetabolomics,which lipidomics,andglycomics technology in developments Recent New advancedtechnology:global“-omics”approach Biosignatures: a set of biomarkers that provide a portrait of the biological condition. outcome in terms of benefit, or harm or lack of benefit or harm. Surrogate biomarkerend-point: that is intended to substitute for a clinical end-point, predicting clinical of terms in function, disease effect, progress, of recovery, outcome survival or final death. the reflects that variable or characteristic end-point: Clinical Surrogate of protection: validated marker of correlate of protection. the individual is being protected against infection developing and/or disease. Correlate of protection: measurable sign in a host in response to an infectious agent processes,indicating pathological whetherprocesses or physiological/pharmacological responses to an intervention. Biomarker: characteristic that is objectively measured and evaluated as an indicator of normal biological Definitions oftermsinbiomarkerresearch point-of-care assays for early diagnosis and treatment of active TB patients in high-incidence validatedofneedtranslatedcountries. biomarkersinarealso beWe run. long ascan thatthe disease the in of transmission of chain the break control programme to the significantly help wouldThis (protection). disease the resistingdevelopmentof of probability highest the have who those and (susceptibility) thatdistinguishcan betweenindividuals infected having thehighest riskofdeveloping TB secondary biomarkersfind to absolute need an thus is There 2007). (Kaufmann,epidemic HIV the by 100-fold latently infected and the risk of developing secondary clinical TB has been compounded at least about (Parrish,23% Dick It &isBishai, further estimated 1998). that one third of the world’s population are ofreactivating the disease and developing secondaryclinical isestimated TB to befrom 2% to about These individuals can remain latently infected and live their life without thisInanysituation, sign of TB. Their lifetime risk effectiveimmuneresponse.infectionan thecontainwith to able are restthe whiledisease primary the tovictimfall 10–12%exposed) those totalofof (a theseof 40% aboutindividualsexposed and priority on the research agenda. from establishing itself, but the actual pathophysiology of this process is still unknown and should be a exposed to these infected aerosols are able to deal with the encounter therebyinfectionthethroughinhalation.individuals sourceof common ofFortunately, most preventing general,70% in the infection the arediseases cavitary open withpatients TB pulmonaryactive by up Infectedcoughedaerosols Natural historyofTBdiseaseandopportunitiesforbiomarkersdiscoveries Infection Biology, Berlin, Germany Dr Shreemanta Parida and Professor Dr Dr h. c. Stefan H. E. Kaufmann, Max-Planck Institute for challenges, prospectsandinterventions A4.7. NewinsightsintopromisingbiomarkersinTB: Source:Kaufmann &Parida,2008;Biomarkersworking group,2001;C Mtb is thoughtis toreside theinhuman subjects non-replicating ain persistent stage. Mtb establishes itself successfully in about 30% of immunocompetent, 2 olburn, 2000. 107 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 108 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 3 lineardiscriminantpatients,ausing clusteranalysiscould TB that discriminationgroup studyoptimal for assessedsubsequentbiomarkersstudy, werecandidate a In TB and healthy control subjects, suggesting its involvement in the diseasefound processesto be (Jacobsen, differentially2005). expressed in the studies conducted by member ourof Ras-associated groupsmall GTPases –on a specific PMBCs regulatorfrom of intracellularpatients vesiclea with trafficking,– was Rab33A 2005). al.,et (Repsilberinformation confoundingselection,biasorcompromised by be and epidemiological studies, need to be applied to microarray experiments and their validity should notclinical bothcomparability,fromprinciplesknownof wellare groups. Therefore,which so-called the study the of choiceappropriate the on rely heavily studiesexpression gene of results and Validity focus in infectious diseases. outcomes.This approach has been successfully used clinicalin the field especially of oncology and currently and isdrugs majora of side-effects efficacy, predict to order in levelgenomic the at drugs effectivelyexploitedinvestigatebe topharmacodynamic ofcan effects the body.Thishuman the in treatment.drug Lymphocyteexpressiongeneclinical, links pathologicaltobiochemical changesand and diseasesbetweenexpression differentialgene ofassessment clinical in sourcetissue feasible applicationsto drugdiscovery anddevelopment. Dueto itsaccessibility, peripheral its isbloodthe most as well interpretation as for analysis data and technologies in advances significant several with years,five last the over rapidly evolved has field cellularlevels.The theproteome andthe genome, part) protein-coding the between link dynamic organism, providing whole a or tissue, cell, organ (mRNA: expression a profiles in transcript gene in changes global evaluates analysisTranscriptome Transcriptomics many host molecules in pathogenesis and protection. either purified proteins or from recombinant expression systems. We have also investigated thestartedwith use of rolecrude preparations of of antigens and has led to the current use of subunit antigens, effectiveinterventionsdesigncontrollingtodisease.This for theas immunological viewpoint,so an Since the time of Robert Koch, the scientific community has struggled to understand the disease from Immunology (Young, Stark & Kirschner, 2008). years to seconds from rangingtimescales disparate over occurringlevel, population the to cellular understand the complex interactions of persistent infection such as TB that range from molecular and betterentiretytothebiologicalscopesystem.3 processesthe alsogivesofa us Thisin thathappen new discoveremergent toproperties that mayarise isfrom theinteraction biology ofsystemsmultiple processes ofinorder to understand goals the of One contexts. of variety a in and biosciences, of insteadthe in usedwidely (integrationbecome hasonwards,term the perspective2000 Particularlyfromthem.study to reduction) new a using thus systems, biological in interactions of complex study systematic the on focuses that field study biological new relatively a is biology Systems and hence the validity of transcriptome profiling results(Jacobsen et al., 2007). improvesinterpretability,heterogeneitycellulargreatlytranscriptomefor analysesdeconfounding of specimensfrequently confoundsanalysis datamicroarrayin studies havewedemonstratedand that tissuecellularcompositionof theHeterogeneity in 2006). al.,et (Jacobsen 33A associatedGTPase noninfected healthy subjects using a minimal group of genes comprising lactoferrin, CD64 and the Ras- http://en.wikipedia.org/wiki/Systems_biology Mtb -infectedhealthysubjectsand 4 body.humanthefoundin be components canfoodthat 3500 and drugs metabolites,1200 characterized 2500 and catalogued Theymetabolome. human the of draft 2007, scientists at the University of Alberta and the University of Calgary, Canada, completed the first currentlypossible to analyse the entire range of metabolites by a single analytical method. In January changing defined notfrom readily second.metabolome secondtoAlthoughbeis theenough, it can transcriptomics and proteomics; like the transcriptome and the proteome, the metabolome issample, such asasingledynamic, organism (Glassbrook &Ryals, Theword 2001). was coined in analogy with hormonesandother signalling molecules, andmetabolites)secondary tofound be within biologicala Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, the physiology of that cell. storyofwhat might happeningbe incell,a metabolic profiling can give an instantaneous snapshot of Thus, while transcriptomicenvironment. the(mRNA gene evenexpression or pathogen, data) and the proteomicor hostanalyses thedo fromnot derivedtell be the wholecan interactions,metabolites host–pathogen In profiles. metabolite small-molecule their of study the specifically, – behind leave Metabolomics is the systematic study of the unique chemical fingerprints that specificMetabolomics cellular processes mass with electrophoresis two-dimensional high-resolution spectrometry, was combiningused to compare the approach,cellular protein composition proteome of two virulent strainsA of & (Wang treatment drug ThisMarcotte, approachparticular can2008). be used to elucidate a novel targets with for new drug associated developments. changes expression protein specific show contrast,systems related tothedrugs’targets, lipid,suchasaminonucleotideand acid metabolism, translation,productionthatdownregulatedcontrol,energyarecycletreatment.andcell drug with In in responses drug anti-TB of analysis Proteome challenge. amounts of target antigens by effective chromatography from sample preparations still poses a major betterwithanalyticaltools. However, abilitythetodeplete abundant proteins toseparateand scanty a contains proteome Serum treasure trove 2007). of protein Veenstra, biomarkers. &More and more proteins Xiao in different (Issaq, biofluids are biomarkers being identified prognostic potential or asdiagnostic used pathological be can the these indicate Hence symptoms. clinical to of onset serve the before even canabnormalities structure and function, abundance, protein in Alterationstime. of point a at cell a by expressedproteins of complementfull the of study the isProteomics Proteomics postgenomic technology for pattern-recognition analyses of biological samples. emerging proving an approach field, which is is tissuesuperiorits and or otherfluid any to anybody spectrometry, of which 41 were identified, including32 mycobacterialspots) attenuatedthethese ofspotswerestrains. total analysed (40 mass 53 Aofby in or spots) (56 virulentthe exclusivelyin wereeitherdetected 96 these, Of proteinspots.distinct 1800 about comprisingpatterns silver-stained in electrophoresis resulted two-dimensional by cells readingopenframesthe in 4000 two attenuated strains of known to be missing in BCG. The remaining 20 spots unique to in deleted be to previously reported genes by proteins encoded as identified were spots http://www.metabolomics.ca M. bovis M. bovis BCG (Mattow et al., 2001). Mtb BCG, in order to identify unique proteins of these strains. From the genome,separationthe proteins of frommycobacterialwhole Mtb were identified as proteins encoded by genes that are not Mtb -specific spots.Of these, 12 . smegmatis M. 4 Metabolomics can be applied toapplied be Metabolomicscan oe sse shows system model Mtb Mtb M. bovis M. -specific -specific with 109 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 110 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted efficaciously although it does not achieve sterile eradicationpathogen thecontrol ofto hand mechanismsat hassystem immunehost the Thus,disease. of signs infectedbecomewith who those all of Africa) sub-Saharan in (approximately80% majority vast the thatargued have We Why developbiomarkers forTB? 2008). multipleplatforms: these wouldmore beappropriate thansinglefora TB biomarker (Jacobsenetal., Biosignatures represent a set of biomarkers in combinations of either multiple markers using single or immune response in TB, using a battery of assays in combination with specific intermediatesmall molecules bymetabolomics profiling. T-cell responses are studied to examine the thebiological for transcriptomics, system using DNA/RNA proteins for proteomics, and enzymes and Using various technology platforms, either alone or in combination, biomarkers can be studied across Types ofbiomarkers Currently, different purified small molecules of approach. this of proof-of-principlesubstantiate theconsistent andwere studies both account,into identified and named, whereas about another235 werebe could130 about whichdifferent of groups, twooutthesedown-regulated between unnamedor eitherup- metabolites. Taking all of thesebe tometabolitesfound factors 365 together,aboutstudieswere there two these Analysing Africa. South latentlyand TB infectedclinicallyand healthy(TST-positive) contacts fromendemicthe population in The study has been repeated in 48 age- and sex-matched individuals with newly diagnosed pulmonary results of prospective studies in other “-omics”. development. An integrated approach is needed, to examine the evidence and to put effectivelythis withother togetherdrugregimens) wellas potentialas withmarkers toscreen candidate novel drugsin drug-resistance markers, for predicting potential non-responders (to manage these groups of patients monitoringchemotherapy,patientsforduringused identification forof beinfection, and TB ofstate tofurtherexplore potentialthe metabolomicsof toidentifynovel biomarkers distinguishthatcan the InitialAfrica.Southpopulationresultsin convincingare enoughTB-endemic healthy fromcontactsa individuals with newly diagnosed pulmonary TB patients and latentlyUniversity, infectedSouth Africa, (TST-positive) using and Metabolon’sclinically platform using sera samples from partner 38 Metabolon age- Inc.,and based sex-matchedin Durham, USA, along with our academic collaboratorsindustrial leading the at collaboration with Stellenbosch in group our by conducted study proof-of-principle first The of the network. with the profiles of transcriptomics, proteomics, lipidomics integrated be andthen would These states. pathological glycomics physiological or specific reflecting perturbation for resolving the complexities the use of combinations of chemometric and mathematical modelling methods to classify biochemical the generated by during these techniques and their inherent lipids multivariate profilingdensitiesinformation andcapabilities High culture. usuallymacrophage proteinsnecessitate in infection of genes,course the over involvingcrosstalk molecular molecules) signalling lipid and ATP cholesterol, glucose,like compounds (e.g.pathways biochemicalintermediatemetabolitesfrom and primary of and host the of state metabolic the monitor to way under are Efforts of metabolites if investigate to platforms spectrometry chromatography-mass gas and spectrometry platforms, to be curated in a database. Mtb can be detected among the unnamed metabolites that were never run on these analytical these on run neverwere thatmetabolites unnamed the amongdetected be can Mtb will control the pathogen throughoutpathogendevelopingcontrolwithoutlifetheirthewillclinical Mtb are being analysed by liquid chromatography-mass Mtb . Will it be possible to learn from this Mtb dpcig h interplay the depicting , Mtb antigens. 6 5 this is the long incubation time of latent would need to be enrolled to get adequate power in the study to obtain conclusive evidence. Added to rate of 1 in 100, as seen in the worst-hit parts of South Africa and Swaziland, largewords, groupsclinical of diagnosisindividuals of TB disease serves as clinical end-point of the trial. Even clinicalwithmeasuredtrialis preventionthe by a outbreak groupstudyactiveparticipants.inof TB other In TB incidence III phase a in vaccine a of success Currently, the adults. in efficacy determiningprotectivevaccine definitelyataimed clinicaltrial III phase development researcha andthepipelinewith of end the at future.greatestnearcomethustheThe willclinicalobstacle expectedentertrialsinare IIIto phase have already completed, clinical phase I trials and a few candidates have reached phase II trials. Some novel TB vaccines and perhaps also with new drugs against TB. Several TB A majorvaccines goal of our strategyare is the definitiondue of ato surrogate enter,end-point for clinicala phase II/III trial with or as the highest to in6.3% Ethiopia 2008). (WHO, to 257 per 100 hard hit by TB and TB HIV/AIDS. incidences range from 940 per 100 immunologicalwhichresponses“Learnprovideprotective immunity”. countriesinvolvedallTheare topics): Challenges Grand defined 14 the of (out 6 Challenge Grand under specifically and themes goal-oriented majorsevenVaccines” among NewCreate “To goal thematic broader the underfalls collaborativenations,ThisUganda.6namely,project and Africa Ethiopia,SouthMalawi,Gambia, the programme,5 we are attempting to define biomarkers forTB in the context of in HIV/AIDS five African fromsupportWith theBill Melinda & Gates Foundation undertheGrand Challenges GlobalinHealth immunity against TB – fall into this definition(Box 2). and susceptibility to natural infection with (Biomarkers working group, 2001). Thus, the biomarkers we aim to identify – biomarkers of protection and evaluated as an indicator of a normal or a pathological process or of the responseIn theto anarea interventionof clinical trials, a biomarker is defined as a characteristic feature that is objectively measured clinical end-point of TB disease outbreak. the to prior efficacy vaccine informationabout valuableprovide could diseaseprotectionagainst of could be defined and serve as a guideline for monitoring vaccine-induced immunity. Such a biomarker exploit their value as potential indicators of protection? If so, then a biomarker of protection against TB naturallyresponsebetterunderstandhostmechanismsinducedthoseandcontrol thatinfection and failure or relapse. reactivation or latent infection; and (iii) predicting drug treatment outcome, i.e. treatment toolssuccess, to moredrug reliably diagnose active prognostic (ii) TB; markersdiagnostic novelof infection outcome,(i) for: i.e. basis TB disease the provide can TB biomarkersfor informationonvalue, added an As extraordinarily helpful. be would schemes prime–boost heterologous in candidates vaccine different of combinations on informationterminationasinto,well decisionsclinicalfororvaccineasentrytrials of,IIabout phase Even before a phase III clinical trial, biomarkers that provide robust information for educated decisions process at different stages of clinical trials. gravesituation,thisofviewacceleratebiomarkers todevelopmentsubjects.Inhelpthestudy could clinical trials for TB vaccines may last years (possibly a decade, if not longer) comprising thousands of time point, with 5–10% probably occurring within the firsttwo years.Thus, it has been estimated that of those who become infected with http://www.gcgh.org/NewVaccines/Challenges/ default.aspx LearnaboutImmunologicalResponses/Pages/ http://www.biomarkers-for-tb.net/

0 00 in00 the Gambia as the lowest, and from 70% HIV infection in TB patients in Malawi Mtb Mtb during a phase III trial in Africa will develop active TB at a later Mtb infection, ranging from months to lifelong. About 10–20% , leading to the adoption of a marker of vaccine-induced

0 00 00 in South Africa as the highest 111 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 112 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted to stop this resilient increaseawareness among donors andto steer the process to collectively andindividually contribute newtools to ultimately eliminate (Feuer, TB The scientific 2007). community has the responsibility to 2 billion per year in order to support the basic, applied, and operational research necessary to develop TreatmentAction Group, research TB and development investments need to increase fivefold to US$ development over the last few decades (Kaufmann & Parida, 2007). According Thepresent to failure theto adequately control latestisthe TB result of lost reportopportunities in funding ofresearch and the intellectual property issues at the outset in clear and stringent terms to move forward progressively. cohesively,essentialinteraction.honesttheworksolve toandistoought open We It with vertical). and (horizontalplatforms or diseasesdifferent forinitiatives similar interactionswith and synergies collate to efforts concerted by initiatives, approach, expertise andexperiences holistic within(synergies) thescientific community. a This also demands using protective addressed of be understanding to basic overall needs an This immunity. have not do still we century, a over efforts Despite Challenges andwayforward Required performancecharacteristicsofbiomarkers Identification/differentiation ofdiseasestate Biomarkers needinthecontextofTB • • • Immune • Treatment • Infected: • Surrogate • • • •

Diagnostic • Simple Sensitive Cost–effectiveness High Easy Surrogate 2 1 3 2 1 2 1 3 2 1 . persisters with risk of relapse . with high risk of disease (susceptibility) . markers of infection . markers for assessing potential drug candidates . drug resistance. . with prognosis of protection . prognostic markers of reactivation/disease. . markers of relapse . special subsets – extrapulmonary TB, paediatric populations, with HIV coinfection. . markers of treatment failure (drug resistance).

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c andidates. probability that the test will give the correct diagnosis: the us thistell not do requires they test, a of thevalue the markersuseful positiveof are specificity andsensitivityand Although negative predictive The authors propose to test the predictive value of the markers they identified in a largerpopulation. however,expression.not,generelapsesdid by They distinguish reinfections.relapsesbetween and differentuseda cross-sectional etdesignal.(2007) Mistryto distinguish betweenrelapses non- and will be obtained from an unsuccessful shortened regimen. effective regimens will not yield useful results when validatingHighly a surrogateMitchison,1999). & Ellardmarker: (Fox, morepopulationAfrican useful the inresults lowerwererates relapseregimen, higher culture be conversion mayrates resultswere observed asin Hong Kong important, compared bewith East Africa will with populationsthepopulation dependent. same Results of from British Medical Research choice Council studies indicated the that whereas surrogate, a of evaluation the In necessarily provide a substitute for long-term relapse rates. alreadyindicated, culture conversion ratesdifferentiate do betweentreatment regimens maynotbut Thisstrategy reduces thenumber oftrials neededrunsbutthe risk ofrejecting effectivean drug.As survival.longer-termtoof forwardtrialstakecandidates tothe select to used progression been has tumour of intermediate end-point The testing. for available agents new of number the in increase There has been a change of approach in the conduct of some recent cancer trials due to the substantial points must be objectively measured and reinfections need to be clearly distinguishedrelapse.End- for fromfollow-upadequate withrelapses. trial a validatedin be to need 2008b) al.,et (Rustomjee – control trial of ofloxacine-containing, short-course regimen formulticentre, therandomized,a treatment – trial OFLOTUB of the pulmonaryin foundregimens tuberculosisvarious inresults counts colony sputum serial in differences impressive The rates. relapse and failure subsequent to respect with It is essential that markers that appear to distinguish between regimens during treatment are validated 2004). followed by a continuation phase with different drugs and differentintensivecommonwas phase a where durations A StudyUnion the in(Jindani, andResearch1981)Councils, Medical Nunn & Enarson, the same drugs in the intensive phase but relapse rates ranged from 11% to 32% (East African/British in the Fourth East African Short course study, where patients in four of the regimens received exactly or in the duration of treatment, these markers may have no predictive value at all.although Thisthey is may wellbe good predictorsillustrated of outcome, if there is variation in the drugs given subsequently whethermicroscopy, culture theorrate ofdecline allof–sufferCFU from theobvious limitation that In the context of anti-TB treatment trials, bacteriological results during the early stages of treatment – Its predictive ability should not depend on the treatment being assessed (Prentice, 1989). require that it be not only a good predictor of treatment outcome but also explain the treatment effect. washigher in the combined treatment arms than inthe monotherapy arm. A perfect surrogate would diseaseprogressionwithproportion the arms,combined thegreater inwas RNA HIV indecline the to some quite unexpected results (Delta diseaseCoordinating Committee and and reduction Virology Group, RNA Although1999). HIV progression, in the Delta between trial – associationcomparing inversezidovudine monotherapy clear with combination a therapy is– gave rise there Whereas drugs. antiretroviraloftrials in chemotherapy,shown been shortening TB haslimitationstheirhave theyas forpotential the with drugs candidateidentify to opportunity the offermarkerssurrogate Although Professor Andrew Nunn, Medical Research Council Clinical Trials Unit, London, England A4.8. Designing studiesforTBandTB/HIVbiomarkers:challengesopportunities 113 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 114 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted would receive an unnecessary extension of their regimen. only specificity80% would mean that, in addition to the 15% requiring extra treatment, a further 17% month regimen required 15% of four-patients a to Ifrelapses. missinghaveavoid extendedto sensitivity treatment, 100%, to a closetest high, withvery 100% a sensitivityhave must but treatment,it their extensionof an needingthose identify tomanagementpatient in used be to biomarkeris a If predicted but half of those predicted to be relapses would be non-relapses. increasesittreatmentrelapse51%. contextto5%rate,thearelapses oftrueaIn all with would be value predictive ofpositive only 2%. With a a prevalence have will of the 3%, predictive 1000 value in is and 38% with 1 a prevalence prevalence of of low 5%, a of context the in specificity 95% and suchas relapse) within the chosen population. To take an extreme case, atest with 100%sensitivity abnormality, (or disease the prevalenceof the on dependentcritically are These test. the of values 115 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 116 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 117 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 118 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 119 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 120 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 121 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 122 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Garbe TR, HiblerNS, Deretic V (1996). 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The New EnglandJournal, 324(12):781–788. ofMedicine American Review Disease,123(2):165–170. ofRespiratory (4-month) chemotherapy tuberculosis. Secondreport of the4thstudy. regimensinpulmonary The Microbiology Reviews, 18(4):687–702. Medicine, 160(1):203–210. as asurrogate for responsetochemotherapy. American Journaland CriticalCare ofRespiratory as surrogates for clinicaloutcome. AIDS, 13(5):565–573. 50(9):3154–3156. pharmacodynamic measuresintuberculosis treatment.Antimicrobial Agents andChemotherapy, against Leishmania major.Nature Medicine,13(7):843–850. more efficientdrug development. Journal ofClinicalPharmacology, 40(12 Pt2):1419–1427. tuberculosis patients.EuropeanJournal ofClinicalMicrobiology&Infectious Diseases,21(1):1–5. 74(1):20–25. monocytes after BCGvaccination inBritishschool children. Immunology, ClinicalandExperimental after vaccination Immunology,9(4):901–907. againsttuberculosis. ClinicalandDiagnosticLaboratory infarction. The New EnglandJournal ofMedicine,327(4):227–233. 12(2):146–151. tuberculosis patients.Thepulmonary InternationalJournal of Tuberculosis andLungDisease, of Society Tropical MedicineandHygiene, 99(12):893–900. in unprocessedurineof Tanzanian patientswithsuspectedtuberculosis. Transactions oftheRoyal Immunology, 36(12):3346–3355. influence on vaccine-induced protectionagainstMycobacterium tuberculosis. EuropeanJournal of randomised clinicaltrial.Lancet,360(9332):528–534. a week for tuberculosis treatmentofdrug-susceptible inHIV-negative pulmonary patients:a gamma. Vaccine, 19(17–19):2298–2302.

TM, Andersen P(2005). Vaccines for tuberculosis: novel conceptsandrecentprogress.Clinical 123 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 124 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Mittrucker Poor HWetal.(2007). correlation betweenBCGvaccination-induced T cellresponsesand Gene-expression Retal(2007). patternsMistry inwholebloodidentifysubjectsatriskfor recurrent Mitchison DA (1993). Assessment ofnew sterilizingdrugs tuberculosis for by treatingpulmonary culture Mattow Jetal.(2001). IdentificationofproteinsfromMycobacterium tuberculosis missinginattenuated tomycobacteria.Martineau Denhancesimmunity AR etal.(2007). A singledoseofvitamin American RecombinantMcShane Hetal.(2004). modified vaccinia virus Ankara expressing antigen85Aboosts Lok Moleculardifferentiation KHetal.(2002). ofMycobacterium tuberculosis strainswithoutIS6110 Kaufmann SHE,Parida SK(2008). 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Immunology, 15(4):638–643. in urineasapossiblemolecularmarkertuberculosis. ofactive Clinicaland pulmonary Vaccine 125 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 126 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted WHO/TDR Reportfor (2007). oftheexpert onimmunotherapeuticinterventions tuberculosis consultation van Lost derGreefJetal.(2006). intranslation?Rolesolvingtranslationalproblems ofmetabolomics emergingscience.In:Boulton –animportant E,ed.Businessbriefing. Ryals Metabolomics J(2004). Parida SK,KaufmannSHE(2008). The questfor biomarkers intuberculosis (submitted). Beecher C(2003). profiling:itsrole The In:Harrigan G,GoodacreR,eds.Metabolic humanmetabolome. Bibliography forAnnex4 and Training in Tropical Diseases(TDR). Geneva,. 2007 29–31January World HealthOrganization/Specialprogrammefor Research in drug discoveryanddevelopment. Drug Discovery Today: Technologies, 3:205–211. PharmaTech. (PharmaTech 2004 series).BusinessBriefingsLtd:51–54. in biomarker discoveryandgene functionanalysis. Kluwer Academic Publishers:311–319. 7 children. All biomarker studies described in this meeting report are applicable to childrennon-infected and HIV-infectedas well.in drugs TB new oftrials clinicalconduct to needgrowing a is There Schaaf, 2007). & Donald children(Coulter,2008; in not but adults intrials clinical testedin beingare drugsanti-TB investment has been TB made in of research progression preventon diagnostics, thereby drugs, and vaccines childand biomarkers a studies. indisease.Newer While aresurgence TB of interest in childhood of over TB the past diagnosisdecade has occurred, verylittle therefore early are an techniques make diagnostic to Better al., needed 2002). etalso al., (Cannas et adults Kafwabulula in 2008; than al., limited et Connell more even 2008; are children for data the developed, being are tests, which take into account recent advances in molecular diagnosticsensitivebiology, and rapidimmunology morenewer andAlthoughproblematic. chromatographyischildren from samples sputum microbiologicalObtainingevidence.without made isdiagnosis clinical a cases most in andchildren in positive be to likely less investigations are these adults, with However, compared 2007). al., et Marais 2007; (Marais, householdchild’s that within caseinfectious an of discoveryfollows usually and testing, microbiological and testing skin tuberculin radiography, chest on based traditionally is childrenin TB diagnosisof The 2002). al.,Chintuet 2003; al.,et picture(Ansari clinicaltheconfuse childrenandtheclinical diagnostic criteria arelonger noapplicable since opportunistic lunginfections in presentationclinicalpicture TB andthe changedof has 2007; CoinfectionHIV with Marais, 2007). al.,etMarais 2008; (Coulter, difficult very proved historically has children in TB of diagnosis The A5.2. DiagnosisofpaediatricTB WHO, 2008). 2006; (WHO, priority global currentlyconsidereda is adults, in children,as in TB andgroup,Illness was dismissed by these studies, which were used by theproblem health publicWHO Integrated a notchildren wasin Management TB perceptionthat The of Childhood 2003). al.,et Ansari 2002; al. et et al., 1993). These results were confirmed by other autopsy studies of children fromdying Botswanaof (Ansari respiratory illnesses in Lusaka, Zambia, found that 24% of landmarkSwaminathan,Maraisal.,etnecropsy2007;ARekha& children2007; 164 of study these 2007). children had died of TB (Chintu childrenmajorclinicalinMarais,Saharana becomenowAfrica,problem TB has al., et(Chintu 1993; sub- in especially TB, with people HIV-infected of number the in riseassociated the and epidemic HIV/AIDS the ofadvent the Withhousehold. theirfrom adult aninfectious commonlyindividual,an from transmission recent indicating community the in event sentinel a represent TB with children worldwide,cases TB all of proportionrelativelysmall a foraccountchildren among cases Although A5.1. Introduction International Health, University College London, London, England Health, London, England, and Professor Alimuddin Zumla, Centre for Infectious Diseases and Professor Nigel Klein, Great Ormond Street Hospital for Sick Children, Institute of Child di Annex 5. Supplementary contribution fromdiscussionsatthe meeting. a gnos ti Pa c s edi a nd biom a tri c

TB a : rkers 7 127 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 128 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted really offer major advances over the TST (Connell et al., 2008). As with adults, there is a need to need a forchildren.theseusedwhenassaysnewshouldandbe if Furthermore,is clarify desperate thereathere is adults, with As 2008). al., et (Connell TST the over advances major offer really QuantiFERON the either if unclear yet as is Itpromising. look data the of somealthough adults,in children thanin clear lesseven isdiagnosticnewer tests the of utility The degree of immunosuppression, particularly the count.CD4 False-positive TST results may also occur. the ondependent is and 10%than higher certainly is it butunknown, is HIV infectedarewith who TB withchildren in TST false-negative of ratesevereinfections. otherThe illnesses,malnutrition or mayTST also occur in children with severe TB disease, those with debilitating or immunosuppressive immunosuppression.toFalse-negativecontribute itself may disease TB thattreatment,suggesting culture-proven with children TB do normal not otherwise react of to 10%tuberculin to initially. Up children. younger Most inof foundthese ischildren sensitivity will lowest become The reactive during (false-positive results). specificity andresults) sensitivity(false-negative poor bothfromsuffers TST disease. tolowerdueusuallynumbers is extrapulmonarymycobacteriaoflessthan50% TB these insites of children with from tissues or fluids body otherfromculture yield infants. The in 70% as high as be consecutiveThreemorninggastric TB.aspirates pulmonary yield Culture of gastric aspirates has provided a more useful method of diagnosis in children with suspected even lower mainly because of the paucibacillary nature of disease at these sites. ratesof detection inmicroscopy from other extrapulmonary samples, such ascerebrospinal fluid, are adults.The comparedin75%with children20%, thansampleslessinisproven with TB pulmonary aspirategastricmicroscopyfromof yield Themorning. the in foodingestion ofto collectedprior be stomachovernightcan swallowoftenwhichtherespiratorywilland in secretions, pooledare which gastric contents via a nasogastric tube. This takes advantage of the fact that infants and youngobtaining childrensputum, early microscopicmorning gastric aspirate do, samples theyare alternativetooften an collected when As bydisease. aspiration progressive and,primary haveof theyovernight sputumbecausenegative often produceexaminationis to unable be may infants and children Young A5.4. Diagnosticsandclinicalbiomarkers be exploredto specifically inneed children. that Biomarkerareas studiesparticular shouldsome be appliedare to therethesechildren, special situations. in yield diagnostic low the to addition In A5.3. SpecialclinicalsituationsinpaediatricTB 5. 4. 3. 2. 1.

2007; WHO, 2006; Chintu2007; WHO, 2006; et al.,1995; Chintu et al.,Chintu, 2004; 2007; Oeltmann et al., 2008). devastating effect in many cases (Lishimpi et al., Luo2002; et al., Rekha1994; & Swaminathan, T the vertebrae, is very debilitating in children. M treatment. characterized by solitary brain lesions, sometimes occurring after commencement of anti-TB sequelae. Tuberculomas of central nervous system infection may occur and are usually reported to be 13%, with approximately half of survivors developing permanent neurological of TB in children and occurs in about 4% of children with TB. The overall mortality has been C 25% of infants and children aged less than four years. E T he combination of extrapulmonary disease in combination with HIV appears to have a B in children who are HIV-infected. xtrapulmonary disease is more common in children than adults, occurring in approximately entral nervous system disease, especially TB meningitis, is the most serious complication usculoskeletal disease, which primarily affects weight-bearing bones and joints, particularly Mtb in 30–50% of cases and may andcases of 30–50% in 

or ELISPOT assays will assays ELISPOT or make a concerted effort to invest funds on this area. mustand Africa, sub-Saharan especiallyin paediatricgrowingTB, problem ofthe of importancethe United Kingdom's Department for InternationalDevelopment,Internationalfor Agency DevelopmentStatesUnited andthe donor TDR, agenciesEU, must The now recognizeTB. diagnosing of method mycobacterial and LAM DNA, proteins are underof detectionway and,the if forsuccessful, tests would Urine-based children.providein a non-invasive TB diagnosing of methods new identify to need 129 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 130 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted Global tuberculosis control: surveillance, planning, financing.WHO Globaltuberculosis planning, (2008). control:surveillance, WHO.Geneva, report2008 WHO Guidancefor (2006). nationaltuberculosis programmesonthemanagementoftuberculosis in Rekha B, Swaminathan Childhoodtuberculosis S(2007). –globalepidemiologyandtheimpactofHIV. Oeltmann Reported JEetal.(2008). childhood tuberculosis treatmentoutcomes,Gaboroneand Diagnostic andmanagementchallengesMarais BJetal.(2007). for childhood tuberculosis intheeraof ChildhoodtuberculosisMarais BJ(2007). –riskassessmentanddiagnosis.South African Medical Luo Cetal.(1994). Humanimmunodeficiencyvirustype-1 infection inZambian children with mouthwashes IdentificationofPneumocystisfrom Lishimpi Ketal.(2002). cariniiDNAinoropharyngeal Kafwabulula Evaluation Metal.(2002). ofPCR-basedmethodsfor thediagnosisoftuberculosis by Donald PR,Schaaf Oldandnew HS(2007). drugs for thetreatmentoftuberculosis inchildren. Paediatric tuberculosis inyoung Diagnosisofpulmonary children.Coulter JB(2008). Annalsof Tropical Paediatrics , Connell TG etal.(2008). A three-way comparisonoftuberculin QuantiFERON-TB skintesting, goldand Chintu C(2007). Tuberculosis andtheHIVvirus co-infection inchildren: managementand challenges. Chintu Co-trimoxazole Cetal.(2004). asprophylaxis againstopportunistic infections inHIV- Chintu Lungdiseasesatnecropsyin Cetal.(2002). African children illnesses:a dyingfromrespiratory Chintu Cetal.(1995). Impactofthehumanimmunodeficiency virustype-1 oncommonpediatric Chintu Cetal.(1993). hypersensitivityreactionsduetothiacetazoneinZambianchildren Cutaneous Cannas Mycobacterium tuberculosisA etal.(2008). DNAdetectioninsolublefractionofurinefrom PathologyAnsari NAetal.(2003). andcausesofdeathinaserieshumanimmunodeficiencyvirus- PathologyAnsari NAetal.(2002). andcausesofdeathinagroup128 predominantly HIV-positive References forAnnex5 World Health Organization. children. Geneva, World HealthOrganization(WHO/HTM/TB/2006.371). Paediatric Reviews Respiratory , 8(2):99–106. 12(2):186–192. Francistown, Botswana, 1998–2002. The InternationalJournal of Tuberculosis andLungDisease, HIV. The Journal ofInfectious Diseases,196(Suppl. 1):S76–S85. Journal, 97(10 Pt2):978–982. Lung Disease,75(2):110–115. tuberculosis: changing seroprevalence andevaluation ofathioacetazone-freeregimen.Tubercle and AIDS children illnesses. , 16:932–934.AIDS dyingofrespiratory Lung Disease,6(8):732–737. identification of mycobacterial DNAinurinesamples.The InternationalJournal of Tuberculosis and ReviewsRespiratory , 8(2):134–141. 28(1):3–12. T-SPOT.TB inchildren. PLoS ONE,3(7):e2624. Paediatric Reviews Respiratory , 8(2):142–147. 364(9448):1865–1871. infected Zambianchildren (CHAP):adouble-blindrandomisedplacebo-controlledtrial.Lancet, descriptive necropsystudy. Lancet,360(9338):985–990. illnesses inZambia.Journal of Tropical Pediatrics, 41:348–353. Childhood, 68:331–334. infected withtuberculosis andthehumanimmunodeficiencyvirus. Archives ofDiseasein 12(2):146–151. tuberculosis patients.Thepulmonary InternationalJournal of Tuberculosis andLungDisease, Disease Journal, 22(1):43–47. positive and-negative pediatricreferral admissionsinBotswana. hospital The Pediatric Infectious 6(1):55–63. patients inBotswana, 1997–1998. The InternationalJournal of Tuberculosis andLungDisease, 131 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted 132 Joint TDR/EC expert consultation on biomarkers in tuberculosis • Not to be reproduced or quoted

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