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PULMONARY PERSPECTIVE

Getting Personal Perspectives on Individualized Treatment Duration in

Multidrug-Resistant and Extensively Drug-Resistant Tuberculosis

Jan Heyckendorf1,2, Ioana D. Olaru1,2, Morten Ruhwald3, and Christoph Lange1,2,4,5

1

Division of Clinical Infectious Diseases, and 2

German Center for Infection Research (DZIF), Research Center Borstel, Borstel,Germany; 3Department of Infectious Disease Immunology, Section for Human Immunology, Statens Serum Institute, Copenhagen,Denmark; 4 International Health/Infectious Diseases, University of L ubeck, L ubeck, Germany; and 5Department of Medicine, University ofNamibia School of Medicine, Windhoek, Namibia

Abstract

Tuberculosis (TB) differs from most other bacterial infectiousdiseases by a very long duration of combination antibiotic therapyrequired to achieve relapse-free cure. Although the standardrecommendedshort-coursetreatment length for TB is 6 months,

the World Health Organization recommends a duration of 20months for the treatment of patients with multidrug-resistant andextensively drug-resistant TB (M/XDR-TB). Apart from the longduration of anti-TB therapy, treatment of M/XDR-TB is veryexpensive and often associated with adverse drug events. Theoptimal duration for treatment of TB likely differs betweenindividuals and depends on a variety of variables, such as the extentof the disease, the immune status of the host, and the virulenceand the drug resistance of the causative strain ofMycobacteriumtuberculosis. Some patients with M/XDR-TBmay have to be treated

with currently available antituberculosis drug regimens for morethan 20 months, whereas much shorter treatment durationsmay be possible to achieve cure for the majority of patients withM/XDR-TB. Personalization of the duration of treatment forTB, especially for patients with M/XDR-TB, would be highlydesired. Until recently there has been little interest in the

identication of biosignatures that could eventually lead toindividual recommendations for the duration of anti-TB therapy.This pulmonary perspective reviews the knowledge on clinical andradiological scores, host- and pathogen diseaserelated proles,molecules, and signatures that are currently explored as biomarkersto personalize the duration of therapy in TB.

Keywords:biomarkers; multidrug-resistant tuberculosis;personalized medicine; treatment duration; extensively drug-resistant tuberculosis

Tuberculosis (TB) is a leading causeof morbidity and mortality worldwide.Although the overall burden of TB has beendeclining at an annual average of 2.2%, inthe past years the number of patients withmultidrug-resistant (MDR)-TB, denedbyin vitrodrug resistance ofMycobacteriumtuberculosis to the two most effective drugsfor TB treatment, rifampicin and isoniazid,and extensively drug-resistant (XDR)-TB,dened as MDR-TB plus in vitro drug

resistance ofM. tuberculosis to amikacin,capreomycin, or kanamycin plus anyuoroquinolone, is dramatically increasing.According to the latest reports by the WorldHealth Organization (WHO), numbers ofpatients identied with M/XDR-TB in theyears 2010 to 2012 were 54,887, 61,907, and83,715, respectively. However, the estimatednumbers of patients with M/XDR-TBare much higher at 450,000 cases, withcorresponding 300,000 patients with

pulmonary MDR-TB in 2012. Moreover,XDR-TB has been reported in 92 countries,and 9.6% of all MDR-TB cases meet theXDR-TB denition (1).

Treatment against M/XDR-TB isrecommended with a combination of at leastfour drugs shown to be effective byin vitrodrug susceptibility testing over a periodof 20 months (2). This is an exceptionaltreatment duration compared with otherinfectious diseases (3). Evidence for this

(Received in original form February 25, 2014; accepted in final form June 18, 2014)Funded by a grant from the German Ministry of Education and Research (Bundesministerium f ur Bildung und Wissenschaft, BMBF) for the German Center ofInfection Research (DZIF) Clinical Tuberculosis Unit (C.L.).

Author Contribut ions: J.H. contributed to the idea, concept, and design of the manuscript; the acquisition, analysis, and interpretation of the data; drafting andrevising of the article; and approved the final version of the draft for publication. I.D.O. contributed to the acquisition and interpretation of the data, drafting andrevising of the article, and approved the final version of the draft for publication. M.R. contributed to the acquisition and interpretation of the data, drafting

and revising of the article, and approved the final version of the draft for publication. C.L. contributed to the idea, concept, and design of the manuscript; theacquisition, analysis, and interpretation of the data; drafting and revising of the article; and approved the final version of the draft for publication.

Correspondence and requests for reprints should be addressed to Christoph Lange, M.D., Division of Clinical Infectious Diseases, German Center for InfectionResearch (DZIF) Clinical Tuberculosis Unit, Research Center Borstel, Parkallee 35, 23845 Borstel, Germany. E-mail:[email protected]

Am J Respir Crit Care Med Vol 190, Iss 4, pp 374383, Aug 15, 2014

Copyright 2014 by the American Thoracic Society

Originally Published in Press as DOI: 10.1164/rccm.201402-0363PPon June 18, 2014

Internet address:www.atsjournals.org

374 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 4 | August 15 2014
mailto:[email protected]://dx.doi.org/10.1164/rccm.201402-0363PPhttp://www.atsjournals.org/http://www.atsjournals.org/http://dx.doi.org/10.1164/rccm.201402-0363PPmailto:[email protected]


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recommendation comes from an individualpatient data metaanalysis of observationaldata from 9,153 patients (4). However,related to the extent of the disease, theimmune status of the host, the drug-resistance pattern, and virulence of the

bacteria, the duration of anti-TB treatmentnecessary to achieve relapse-free cure ishighly variable (Figure 1). This leaves the20-month treatment recommendation forpatients with M/XDR-TB as an averageestimate, which is likely incorrect for thegreat majority of individual patients. Theextent of the variation has recently beendemonstrated, when a 9-month short-course drug regimen for the treatment ofMDR-TB was shown to be highly effectiveto achieve successful treatment outcomein a study from Bangladesh, a country

with low levels of

uoroquinolone drugresistance ofM. tuberculosis (5). TheSTREAM trial aims to further validate this

promising approach in Ethiopia, Republicof South Africa, and Vietnam (6).

A shorter duration of treatment mightalso be used in children with limited MDR-TB disease, where 12 to 15 months oftherapy might be sufcient to attain cure,

whereas in children with extensive diseasea treatment duration of 18 months afterculture conversion would be necessary (7).A recently published study showed thatmore than 90% of children with MDR-TBreceiving a median duration of therapy of13 months had a favorable outcome (8).This would support individualizing MDR-TB therapy according to disease severity.

Although the current WHO treatmentguidelines suggest the adaption of treatmentduration based on a patients bacteriologicalstatus or other markers for treatment

progress,

there is no guidance concerningthe consequences of these markers, and themeaning of other markers for treatment

progress is not explained. Due to theimportant inuence of host and pathogenvariables to achieve relapse-free cure withcurrently available medications and theoverall long duration of anti-TB treatmentof M/XDR-TB, individualization of

the duration of M/XDR-TB treatmentwould be highly desirable. In additionto improvements in cost-effectiveness,individualized treatment regimens achievehigher success rates than standardizedtreatment regimens (9) and may possiblylead to better quality of life. However, theonly biomarker validated to guide clinicianson decisions for the duration of therapyin any bacterial infection to date is theprocalcitonin level in community-acquiredpneumonia (10).

The development of clinical and

radiological scores and identi

cation ofhost- and pathogen diseaserelated proles,molecules, and signatures to monitor theeffect of anti-TB therapy is rapidly evolving.The discovery of biomarkers to guideclinicians for the duration of M/XDR-TBtreatment will have far-reaching clinicaland economic consequences.

This perspective elucidates theexpanding eld of TB biomarker researchfor therapy response and introducespotential future approaches for individuallytailored TB treatment durations, especiallyneeded for the management of M/XDR-TB.

Pathogen-related Markers

Microscopy and Culture

In the early course of anti-TB treatment, themost frequently used method to evaluatea patients state of infectiousness is theidentication of acid-fast bacilli by sputumsmear microscopy (11). As a measure fortreatment outcome, culture conversion(CC) is part of WHO outcome criteria,which have been revised for M/XDR-TB

recently (12). The time to CC (TCC)has also been evaluated as a marker fortreatment outcome (1316) (Table 1).In four different studies with patientswith MDR-TB under second-line drugtreatment, the median TCCs were reportedbetween 62 and 152 days (1317) comparedwith representative 78 days in patients withdrug-susceptible TB (17). The median timeto smear conversion (SC) was 83 days inpatients with MDR-TB (16) and 23 daysin drug-susceptible TB (18), with similardisease characteristics affecting TCC and

treatment start

0

threshold

pan drug-susceptible TB

BIOMARKER

LEVEL

FACTORS INFLUENCING DURATION OF TB THERAPYdisease severity, hosts immune status & genetic background,

bacterial virulence & drug-resistance, availability & quality of therapy

TREATMENTREGIMEN

DURATION OF TREATMENT (MONTHS)

6 12 18 20

TB meningitis

M/XDR-TB

rifampicin-resistantTB

TREATMENT PHASE

INTENSIVE

CONTINUATION

BIOMARKER-GUIDED

TREATMENT DURATION

INCREASING

DURATION OFTHERAPY

TREATMENT FAILURE

Figure 1. Hypothetical model of biosignatures to individualize the duration of antituberculosis

(anti-TB) therapy. The individual duration of anti-TB therapy to achieve relapse-free cure varies

substantially among patients with tuberculosis (TB). Factors that influence the duration of therapy

in TB are the severity of the disease, the immune status and genetic background of the host, the

virulence and drug resistance of the bacteria, and the availability and quality of anti-TB therapy.

Biomarkers related to bacterial load, clinical symptoms, and immune activation change with different

kinetics in individual patients during the course of anti-TB therapy. Currently, fixed treatment durations

(lower panel) with an intensive treatment phase (dark gray) and a continuation treatment phase

(light gray) are recommended for different manifestations of TB and different levels ofMycobacterium

tuberculosisdrug resistance. Biosignatures indicating cure (green lines) or treatment failure (blue line)

could provide information to guide physicians on the decisions for anti-TB treatment duration in

the future. M/XDR = multidrug-resistant/extensively drug-resistant. (Illustration with support of Karen

Smith Korsholm/SCILL, Copenhagen, Denmark.)


Pulmonary Perspective 375


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Table 1. Candidate Biomarkers to Monitor Treatment Responses during Antituberculosis Therapy

Marker Classication MaterialEvaluated inM/XDR-TB Remarks Reference

Pathogen-related markersTCC Time until a patient is considered

culture negativeSputum Yes Evaluated for survival prediction,

treatment response, and failure13, 16, 17

Whole blood bactericidal activity Bactericidal activity againstMycobacterium tuberculosisin whole blood culture duringTB treatment

Whole blood No Marker for treatment response 91

TTD Time until a culture is positivein days

Sputum Yes MDR-TB with delayed TTD, treatmentresponse

2023

TB-RNA Quantication of mycobacterialRNA

Sputum No Treatment response 31, 37

TB-DNA Quantication of mycobacterialDNA

Sputum, urine Not specied Treatment response 32, 35, 36, 38

TB-antigens Quantication of mycobacterialantigens

Sputum, urine,serum

Not specied Treatment response 2430

Host-related markersClinical items

Clinical score Changes of patientcharacteristics in thecourse of treatment

Clinical scoreitems

Not specied Treatment response 39, 40

Radiological parametersChest radiograph score Change of TB correlates

with treatmentChest X-rays Yes Outcome predicti on and treatment

response4143

Chest CT scan TB correlates with TTD CT scans Single drug-resistant, noM/XDR-TB

Longer TTD in patients withcavitary disease

42

18F-FDG PET Decline of SUVmax 18F-FDG PET Not specied Treatment response 4447Chemokines, cytokines, proteins, and peptides

Acute-phase proteins/peptides PCT, CRP, sTREM-1, hemeoxygenase-1, sICAM-1,suPAR, sLAG-3,granzyme B, sTNFR I,and sTNFR II

Serum/plasma No Therapy response and therapyoutcome prediction

55, 92, 93

Cytokines andchemokines

Among others IFN-g, TNF-a,IL-8, IL-6, IP-10, VEGF,IL-10, MIP-1a, IL-13 andsCD40L, MCP-1

Sputum, serum,plasma

No or notspecied

Therapy response and therapyoutcome prediction, correlationwith bacterial markers

51, 54, 94, 95

Nutritional markers Adiponectin, leptin, fetuin-A,and retinol-binding protein

Plasma Not specied Severity of disease and treatmentresponse

96

Release assays and specic cellular responsesImmunophenotype of specic

cellsDifferent cytokine responses

of antigen-specic T cellsand frequency changesof these cells

Whole bloodand PBMC

Some studies Therapy response, correlation withpathogen-related markers andoutcome

63, 68, 71, 97

Cell populationsImmune cells without specic

stimulationExpression of characterizing

surface markers (i.e., CD25and CD127 for Treg)

Whole bloodand PBMC

Some studies Disease severity and treatmentresponse

71, 73, 98

AntibodiesCirculating antibodies against

TB antigensHigh throughput proof of TB

antigens (i.e., antibodiesagainst ESAT-6, CFP-10)

Serum No Treatment response 99

Proteomics and transcriptomics and gene expressionGene-expression patterns Different signatures for

distinct disease statusTotal RNA No Treatment response 83, 84, 86, 87

Single or small number geneexpression changes

Candidate gene-expressionchanges

RNA No Treatment response 100

Protein patterns Treatment response signatures Serumproteins

No Treatment response 80, 81

Mass spectrometryVOC Proof of mycobacterial

substancesBreath No Treatment response 7678

Metabolomic patterns Changes of metabolicpatterns

Urine No Treatment response 75

Drug levels and drug activityTB drug activity or

plasma concentrationIndividual drug activity Plasma No Different concentrations/activity predicts

outcome101

Definition of abbreviations: CFP-10 = 10-kDa-culture-filtrate-antigen; CRP = C-reactive protein; ESAT-6 = early secretory antigenic target-6;IP-10 = IFN-ginducible protein 10; MCP-1 = monocyte chemotactic protein-1; MDR = multidrug-resistant; MIP-1a = macrophage inflammatory proteins1alpha; PCT = procalcitonin; PBMC = peripheral blood mononuclear cells; sCD40L = suluble cluster of differentiation 40 ligand; sLAG-3 = solublelymphocyte activation gene-3 protein; sICAM-1 = soluble intercellular adhesion molecule-1; sTNFR = soluble tumor-necrosis factor receptor; sTREM-1 =soluble triggering receptor expressed on myeloid cells-1; suPAR = soluble urokinase-type plasminogen activator receptor; SUVmax = maximum standardizeduptake value; TB = tuberculosis; TCC = time to culture conversion; TNF = tumor-necrosis factor; TTD = time to detection; VEGF = vascular endothelial growthfactor; VOC = volatile organic compounds; XDR = extensively drug-resistant; 18F-FDG PET = fluorodeoxyglucose positron emission tomography.




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SC. However, culture- and smear-basedmarkers cannot serve as indicators forpositive therapy response after CC, and thesensitivities for both 2-month smear (24%)and culture (40%) results to predict relapsewere shown to be low (19). In the above-

mentioned studies, CC ranged from 62 to152 days, representing a relatively shortduration of time in contrast to the entirelength of therapy and leading to a gapof about 15 months between the lastpositive culture and treatment end, asrecommended by the WHO. The declineof colony-forming units on solid mediacultures or the time to culture positivity inliquid media in the early phase of treatmenthas been developed to assess the earlybactericidal activity of a single drug ora combination of drugs (20, 21). Time

to culture positivity has already beendescribed as marker for treatmentresponse in patients with drug-susceptibleTB (22, 23). Possibly, analysis of serialmeasurements of the time to culturepositivity during the early phase oftreatment in patients with M/XDR-TBmight help predict the total time needed forindividual relapse-free cure; however, atthis point in time this remains hypothetical.Importantly, patients with MDR-TB notachieving CC are more likely to fail therapy(13), and reverting of cultures to positiveafter CC or failure to achieve CC may be

a strong predictor for treatment failure.

Monitoring Antigens of

M. tuberculosis during Treatment

During infection, M. tuberculosisderivedantigens are released from the site ofinfection. These are available for directidentication in sputum, blood, urine, orexhaled breath and have been evaluatedfor diagnostic purposes (24, 25) and couldpotentially be used also for monitoringof TB treatment responses (2628) (Table

1). Limitations to this approach are lowlevels of antigens available, necessitatinghighly sensitive assays. At present,mycobacterial lipoarabinomannan (LAM)remains the only explored M. tuberculosisantigen with sufcient specicity forclinical use (24, 25). Concentrations ofLAM in urine specimens of patients withactive TB correlate with sputum bacillaryload (29). However, the sensitivity of theLAM urine assays in patients with activeTB is too low to suggest that this target isa promising marker to monitor treatment

(24), especially in HIV-uninfectedpatients (30).

To avoid delays that occur whentherapy response assessments dependon M. tuberculosis growth in cultures,quantication ofM. tuberculosisspecic

nucleic acids by amplication methods area more rapid alternative for treatmentmonitoring. Results can be available withinhours. Whereas M. tuberculosisspecicDNA can remain detectable also longafter sterile cure, the identication ofM. tuberculosisspecic RNA products,which are shorter lived, is a promisingmethod to identify viable bacteria (31).Another interesting approach includestreating samples with propidium monoazide,which can bind the DNA from nonviablebacteria and prevent it from being amplied

by polymerase chain reaction; thus onlyviable organisms are detected (32).

Monitoring Nucleic Acids of

M. tuberculosis during TreatmentIn principle, M. tuberculosisspecicnucleic acids can be isolated from differentcompartments, such as sputum and urine(33, 34). However, in a recent directcomparison quantication of sputumM. tuberculosis, DNA was inferior comparedwith culture-guided methods to assess TBtreatment responses (35). The reduction ofdetectable M. tuberculosis DNA measured

by the Xpert MTB/RIF was too poor inspecicity to serve as reliable marker fortreatment response monitoring (36). Incontrast, RNA is a marker for viablebacteria, and the decline of quantiablemycobacterial RNA in sputum specimenof patients with TB under treatment hasrecently been introduced as promisingmarker to monitor early treatmentresponses in an early bactericidal activitystudy (37) (Table 1). Monitoring theexcretion ofM. tuberculosisspecic nucleicacids in the urine of patients with TB after

treatment initiation could serve as a markerfor bacterial killing (38) and may haveadvantages over sputum analyses fortreatment monitoring.

With currently available technologies,all pathogen-related markers for TB therapyresponses have a short detection phase inrelation to the total recommended durationof anti-TB treatment. Pathogen-relatedmarkers might be better suitable to identifytreatment failures and relapses (31, 37)rather than to serve as markers of successfultreatment outcome or the time for

treatment duration. As the risk for relapseis substantially increased when treatmentis discontinued at the time when bacteriaare not directly or indirectly identied bycurrently available methods, host-relatedmarkers could be more appropriate to

identify the individual duration of anti-TBtherapy.

Host Markers

Clinical Scores

A simple way to evaluate patient response totreatment is the observation of changes ingeneral clinical characteristics of patientswith TB. Low-cost clinical scoring systemshave been developed to predict mortalityand treatment failure and to monitortreatment response (39, 40). The valueof such scores depends on baselinecharacteristics of the patients with TB,the clinical setting, and the training,commitment, and accuracy of the medicalstaff recording the data. In contrast topatients with minimal disease, people withadvanced stages of the disease should havethe highest probability for changes inthe clinical score on positive treatmentresponses. Although studies are ongoing tocompare clinical scores in patients withdifferent levels of drug-resistant TB, it isspeculative at this moment whether slower

bacterial clearance in M/XDR-TB is alsorelated to the kinetic of clinical scoresbefore and after culture conversion,especially in the continuation phaseof the treatment.

Clinical scores, although appealing dueto theirsimplicity and low cost, are likely notsensitive enough to indicate the end oftreatment and are subject to a high degreeof inter- and intraobserver variability.However, it should be explored whethera combination of a clinical score withbacterial/host-derived markers or results

of imaging studies may be able to serveas instruments for therapy monitoring.

Imaging

Conventional chest X-rays and computedtomography (CT) scans are used to assessthe extent of disease before treatmentinitiation and at intervals in the course oftreatment (41, 42) (Table 1). A simplenumerical score describing the extent ofpathological changes on conventional chestradiographs has been suggested by Ralph andcolleagues (41). However, radiological




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scoring systems may not be useful tomonitor therapy-associated changes inpatients with MDR-TB. A recent study withSouth African patients with MDR-TB couldnot show a signicant correlation ofconsolidations or cavitation in baseline

radiographs with time to culture positivity(43). A highly sensitive imaging methodthat is able to show viable mycobacteriawould be ideal to determine the duration ofanti-TB therapy. CT scans are a candidateas treatment-monitoring tools because theinitial number of cavities in CT scans hasbeen shown to correlate with increaseof time to positivity in liquid cultureunder treatment (42). Alternatively, 18F-uorodeoxyglucose positron emissiontomography (FDG-PET) CT has beenexplored as a method to monitor treatment

responses in TB in animal models as well asin humans (4447). 18F-FDG PET CT wasdirectly compared with microbiologicalmethods and was able to identifybactericidal activity of drug regimens inmice (45). Findings in a rabbit modelcharacterized the behavior of TB lesionsunder treatment with rifampicin orisoniazid (46). 18F-FDG PET CT was ableto indicate for therapy response by thedecline of maximum standardized uptakevalues of TB lesions (44). Still, the tracersuptake properties in scar tissue in contrastto active TB lesions have to be elucidated to

further characterize the diseases behaviorand to derive specic signs for treatmentresponse. As an alternative to 18F-FDG PETCTmediated imaging, other radiotracerscould indicate ongoing inammatory sitesof infection but are not applicable forlarge trials yet (48, 49). Costs and lack ofaccessibility obviously limit the use of thesesophisticated techniques in high-burdencountries.

Immunological Markers

Cytokines and proteins. Inammatory statesof patients with active disease can bemonitored by analyses of peptides andcytokines in blood specimens duringtreatment (Table 1). Treatment initiationresults in a rapid killing of susceptiblemycobacteria and increase in the releaseofM. tuberculosis antigens (50). This willtransiently augment the activity in thealready primed adaptive immune system,resulting in increased cell-mediatedimmune activity, again driving release ofnonspecic mediators such as acute-phase

reactants. Providing that these signaturesnormalize in the course of treatment, thekinetics of such markers may indicatetherapy response and could help cliniciansto better evaluate and assess individualtreatment in patients with M/XDR-TB

(Figure 1). Recently, plasma specimensof 42 patients with TB were investigatedweekly over a total period of 18 months(51). Among 24 measured cytokines,IFN-ginducible protein 10 and vascularendothelial growth factor levels were theonly candidate markers that showedsignicant changes in the course oftreatment in HIV-positive and -negativepatients. In fact, vascular endothelialgrowth factor levels measured after 2 weeksof therapy correlated with the time ofsputum smear conversion. Thesendings

require validation in larger prospectivecohorts and especially for patients withM/XDR-TB. There are numerous proteinbiomarker candidates described in theliterature with different potential fortherapy response as surrogate endpoint(5255). Up to now, no single marker orpanel of markers is recommended forclinical routine use as a biomarker to guidethe duration of TB therapy. Some markershave already been explored in patients withimmunosuppression (i.e., HIV infection)(51), but the hampered immune responsesof these patients could limit the use of

these markers. Still, protein markers aremethodically attractive because point-of-caretests can be easily developed (i.e., lateralow-test platforms) and specimens are easyto obtain (i.e., capillary blood).

Antigen-specic immune responses

and immune-cell phenotypes. The mostaccurately described methods for identifyingM. tuberculosisspecic immune responsesare the tuberculin skin test and IFN-grelease assay (IGRA). Presence of a positivetuberculin skin test or IGRA result deneslatent infection with M. tuberculosis

in the absence of active TB (56). Inroutine clinical practice, ELISA IGRAs(QuantiFERON-TB Gold In-Tube test;Qiagen, Hilden, Germany) or ELISPOTIGRAs (T-Spot.TB test; OxfordImmunotec, Abingdon, UK) are used toevaluate sensitization to M. tuberculosisantigens (early secretory antigenic target 6[ESAT-6] and 10-kD culture ltrate antigen[CFP-10]), which is indicated by the releaseof IFN-gof specic effector T cells (57, 58).Prospective evaluations of IGRA changesover time have not shown promise for use

as monitoring tools in active TB; IGRAsshow substantial intraindividual variationand do not correlate with clinical endpointssuch as smear or culture conversion (59).

Identication of up-regulation ofstress-specic antigens caused by effective

TB treatment could be an attractive methodto identify treatment responses in TB.The IFN-g response of antigen-specicT cells from patients with TB andhousehold contacts to different phase-specic antigens, including 24 antigensof starvation and stress phases ofM. tuberculosis, were analyzed (60). Therewas no difference of IFN-g responsescomparing patients with active TB andhousehold contacts. Cellular responses of1,247 patients with TB to 23 disease-specicantigens revealed substantial heterogeneity

of IFN-g responses due to differentdisease states, HIV status, and geographicalbackground (61). Using disease stagespecictargets may improve cytokine releaseassays as tools to monitor M/XDR-TBtreatment responses (62).

Recently, IFN-g production byM. tuberculosisspecic CD81 T lymphocyteshas been found to decrease signicantlyduring the rst 6 months of TB treatment(63). Still, compared with other cytokinesthat are released by antigen-specic cells inresponse to infection with M. tuberculosis,IFN-g alone may not be the best cytokine

marker to evaluate therapy responses inTB (64). For the discrimination betweenactive TB and latent TB infection (LTBI),secretion of TNF-a by antigen-specicCD4 T cells detected using intracellularcytokine-staininguorescence-activatedcell sorting (FACS-ICS) was shown tobe superior to IFN-g (65). One studysuggested that the frequency of single TNF-a1CD41 T cells with effector memory

phenotype (CD45RA2CCR72CD1272)can discriminate between active TB andLTBI with a very high sensitivity of up to

99% (66). IL-2 is another cytokine that hasbeen explored as a marker for treatmentresponses in TB (67, 68). During thecourse of TB treatment, a shift frompredominantly IFN-gproducing antigen-specic T cells toward predominantly IL-2producing antigen-specic T cells can beobserved, suggestive of shift from effectorto central-memory T-cell dominance andinfection control (68). Along these lines,it was shown that a low frequency ofIFN-g/IL-2 positive CD41 T cells inresponse to puried protein derivative




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has been shown indicative for active TB(67). Although data are still limited,continuous follow-up of patients forFACS of antigen-specic T cells appearsto be a promising method to monitortreatment responses and warrants further

exploration. FACS methods are able tocharacterize more complex and detailedanalysis of immune cell phenotypescompared with IGRAs. However, as onlyfewM. tuberculosisspecic T cells areavailable for interrogation from wholeblood, the FACS methods remain difcultto standardize and perform with adequatereproducibility (69). Fluorescence-immunospot is an emerging simplertechnology that allows for two-cytokineread-out of antigen-specic T cells. Thisassay has shown promise as marker for

pathogen burden (70).Rare immune-cell populations(i.e., gdT cells, mucosa-associated invariantT cells, and exhaustion T cells) seemattractive for treatment monitoring ofM/XDR-TB (7173). Programmed-death-1(PD-1, CD279) is a cell-surface receptorof the immunoglobulin superfamily foundon immune effector cells and is a markeron exhaustion T cells, which play in chronicinammatory states. PD-11 and its ligandsare expressed on T cells, monocytes, andB cells in patients with active TB, andexpression on these cells declines during TB

therapy (71). Theex vivo blockade of PD-1rescues M. tuberculosisspecic IFN-gproducing T cells from undergoingapoptosis (71).

Circulating regulatory T cells (Treg,CD41CD25high1CD1272FoxP31) havebeen evaluated before and 6 monthsafter the resection of cavitary pulmonarylesions in patients with MDR-TB (74).Compared with healthy control subjects,there were higher frequencies of Tregsbefore surgery with a signicant declineto levels, which were comparable to

healthy control subjects at 6 monthsafter surgery.

X-Omics

High-throughput analysis of metabolites,proteins, or transcription productsfrom blood products, urine, or sputumcan provide disease stagespecicmolecular patterns, so-called -omics(i.e., metabolomics, proteinomics,transcriptomics, or mass spectrometry)to unravel previously unidentiedbiosignatures.

Metabolomics analysis using massspectrometry was recently applied tourine samples from patients undergoingtreatment for TB (75). A 23 small molecularmetabolitebased TB early treatmentresponse biosignature was shown to

consistently change in abundance betweenbaseline and across the treatment period(75). This signature strongly differentiatestreatment effect early, suggesting a clinicallyuseful measure for treatment effect from anattractive sample.

Volatile organic compounds (VOCs) inexhaled breath specimens are currentlybeing explored as screening techniques foractive pulmonary TB (76). Comparable toan alcohol breath test, minute amountsof aromatic compounds and fatty-acidderivatives (i.e., camphor, methyl

butenolide phenol, and methyl dimethylbenzoate), specic for M. tuberculosis, canbe traced by analytic detectors (77, 78).VOC detection technologies have not beendeveloped beyond the stage of prototypesand not been validated to monitor thecourse of TB treatment. At present it seemsunlikely that VOCs will be present in largeenough amounts in the exhaled breathafter culture conversion to determinethe end of TB treatment accurately (79).Although the complexity of the currentavailable detection methods restricts eldapplicability, the identication of VOC,

urine, or plasma biosignatures has shownpromise for next-generation TB diagnosticsand treatment monitoring (Table 1).

Aptamer proteomic approachesdiscovered serum signatures related to hostdefense, acute-phase response, and patternrecognition mechanisms in patients ontreatment for drug-susceptible TB (80, 81).Expression levels of acute-phase proteins(e.g., C-reactive protein) changed mostprominently during the rst 8 weeks ofanti-TB treatment (81). Using a similarapproach, the 8-weekM. tuberculosis

culture status could be predicted bya treatment response biosignatureconsisting ofve serum proteins involvedin the coagulation cascade, neutrophilactivity, immunity, inammation, andtissue remodelling with a sensitivity of 95%and a specicity of 90%, respectively (80).

For transcriptomics approaches, RNAarrays methods are capable to assess up-and down-regulation of total RNA of allcirculating peripheral blood cells. In alarge African cohort, active TB could bedifferentiated from LTBI in HIV-positive

and -negative patients by RNA expressionproles (82). A neutrophilic-driven, IFN-ginducible 393-transcript signaturespecic for active TB, which could becorrelated with radiological extent, has beenrevealed by a large-scale total-RNA array

method (83). Besides mainly IFN-gdrivensignatures, there were Fc-Gamma receptorand type-I IFN networks discovered tobe the main responsive systems in activeTB (84, 85). Importantly, changes inthe gene-expression proles could bedemonstrated after the initiation of TBtherapy (86, 87). In these studies (83, 86),the individual changes and the transcriptsignature after treatment were very muchcomparable to the ones of healthy controlsubjects or patients with LTBI. Signatureexpressions showed very little change

between 6 months (therapy end) andfollow-up after 12 months in patients withpandrug-susceptible TB, indicating fora robust cure signature for the time offollow-up. Although gene expressionsignatures are promising tools to monitortreatment responses in TB, they have notyet been evaluated for this purpose.

Conclusions

Although substantial advances have beenmade to characterize the path from disease

to cure in individual patients, we stilluse standardized treatment regimen anddurations for the therapy of patients withmycobacterial infections. Recently there isan emerging interest to explore biomarkersand biosignatures that could guide cliniciansto determine the duration of TB therapyfor individual patients. Candidate markersare still in preclinical or early clinicalevaluation, and none of these markerscan be recommended at present for usein routine clinical practice. Such markersare urgently needed, especially for the

management of patients with M/XDR-TB,where treatment responses are highlyvariable and current recommendations fortreatment durations may not be adequatefor the majority of affected individuals (88).At present, treatment against M/XDR-TBis characterized by a high frequency ofadverse events, high costs, and a poorprognosis. Individualization of the durationof M/XDR-TB treatment, rather thanstandardized therapy, could become ofglobal relevance for the management ofM/XDR-TB if it improves quality of life,




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proves to be cost effective, and ensuresa better treatment outcome (88).

In contrast to clinical trials, wheredifferent stages of evaluation providea possible clue when new drugs may becomeavailable for clinical use, the development

process for new diagnostic methods is lesspredictable. However, the resurrectedinterest in TB drug trials provides excellentopportunities to link therapeutic and

diagnostic evaluations and to studybiomarkers and biosignatures that couldidentify the time-point of relapse-free cureindependent of comorbidities, immunestatus, and pathogen virulence (89, 90). Thevalidation process of such markers in large

prospective cohorts with biomarker-guidedtherapy duration implies long-term closeobservation follow-up periods. Still,individualizing the duration of TB therapy

could be a great step forward for everypatient in the ght against TB. n

Author disclosuresare available with the textof this article at www.atsjournals.org.

Acknowledgment: The authors thank theGerman Center for Infection Research (DZIF) forfinancial support for this work and Karen SmithKorsholm, Ph.D., Statens Serum Institut,Denmark for the artwork of the figure.

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