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 2German Center for Infection Research (DZIF), Research Center Borstel, Borstel,
Germany; 3Department of Infectious Disease Immunology, Section for Human Immunology, Statens Serum Institute, Copenhagen,
Denmark; 4International Health/Infectious Diseases, University of Lubeck,

Lubeck,

Germany; and 5Department of Medicine, University of

Namibia School of Medicine, Windhoek, Namibia

Abstract
Tuberculosis (TB) differs from most other bacterial infectious
diseases by a very long duration of combination antibiotic therapy
required to achieve relapse-free cure. Although the standard
recommended short-course treatment length for TB is 6 months,
the World Health Organization recommends a duration of 20
months for the treatment of patients with multidrug-resistant and
extensively drug-resistant TB (M/XDR-TB). Apart from the long
duration of anti-TB therapy, treatment of M/XDR-TB is very
expensive and often associated with adverse drug events. The
optimal duration for treatment of TB likely differs between
individuals and depends on a variety of variables, such as the extent
of the disease, the immune status of the host, and the virulence
and the drug resistance of the causative strain of Mycobacterium
tuberculosis. Some patients with M/XDR-TB may have to be treated

Tuberculosis (TB) is a leading cause

of morbidity and mortality worldwide.
Although the overall burden of TB has been
declining at an annual average of 2.2%, in
the past years the number of patients with
multidrug-resistant (MDR)-TB, dened
by in vitro drug resistance of Mycobacterium
tuberculosis to the two most effective drugs
for TB treatment, rifampicin and isoniazid,
and extensively drug-resistant (XDR)-TB,
dened as MDR-TB plus in vitro drug

with currently available antituberculosis drug regimens for more

than 20 months, whereas much shorter treatment durations
may be possible to achieve cure for the majority of patients with
M/XDR-TB. Personalization of the duration of treatment for
TB, especially for patients with M/XDR-TB, would be highly
desired. Until recently there has been little interest in the
identication of biosignatures that could eventually lead to
individual recommendations for the duration of anti-TB therapy.
This pulmonary perspective reviews the knowledge on clinical and
radiological scores, host- and pathogen diseaserelated proles,
molecules, and signatures that are currently explored as biomarkers
to personalize the duration of therapy in TB.
Keywords: biomarkers; multidrug-resistant tuberculosis;

personalized medicine; treatment duration; extensively drugresistant tuberculosis

resistance of M. tuberculosis to amikacin,

capreomycin, or kanamycin plus any
uoroquinolone, is dramatically increasing.
According to the latest reports by the World
Health Organization (WHO), numbers of
patients identied with M/XDR-TB in the
years 2010 to 2012 were 54,887, 61,907, and
83,715, respectively. However, the estimated
numbers of patients with M/XDR-TB
are much higher at 450,000 cases, with
corresponding 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 the
XDR-TB denition (1).
Treatment against M/XDR-TB is
recommended with a combination of at least
four drugs shown to be effective by in vitro
drug susceptibility testing over a period
of 20 months (2). This is an exceptional
treatment duration compared with other
infectious 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 fur
Bildung und Wissenschaft, BMBF) for the German Center of
Infection Research (DZIF) Clinical Tuberculosis Unit (C.L.).
Author Contributions: J.H. contributed to the idea, concept, and design of the manuscript; the acquisition, analysis, and interpretation of the data; drafting and
revising 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 and
revising 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; the
acquisition, 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 Infection
Research (DZIF) Clinical Tuberculosis Unit, Research Center Borstel, Parkallee 35, 23845 Borstel, Germany. E-mail: clange@fz-borstel.de
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.201402-0363PP on June 18, 2014
Internet address: www.atsjournals.org

374

American Journal of Respiratory and Critical Care Medicine Volume 190 Number 4 | August 15 2014

PULMONARY PERSPECTIVE
recommendation comes from an individual
patient data metaanalysis of observational
data from 9,153 patients (4). However,
related to the extent of the disease, the
immune status of the host, the drugresistance pattern, and virulence of the
bacteria, the duration of anti-TB treatment
necessary to achieve relapse-free cure is
highly variable (Figure 1). This leaves the
20-month treatment recommendation for
patients with M/XDR-TB as an average
estimate, which is likely incorrect for the
great majority of individual patients. The
extent of the variation has recently been
demonstrated, when a 9-month shortcourse drug regimen for the treatment of
MDR-TB was shown to be highly effective
to achieve successful treatment outcome
in a study from Bangladesh, a country
with low levels of uoroquinolone drug
resistance of M. tuberculosis (5). The
STREAM trial aims to further validate this

promising approach in Ethiopia, Republic

of South Africa, and Vietnam (6).
A shorter duration of treatment might
also be used in children with limited MDRTB disease, where 12 to 15 months of
therapy might be sufcient to attain cure,
whereas in children with extensive disease
a treatment duration of 18 months after
culture conversion would be necessary (7).
A recently published study showed that
more than 90% of children with MDR-TB
receiving a median duration of therapy of
13 months had a favorable outcome (8).
This would support individualizing MDRTB therapy according to disease severity.
Although the current WHO treatment
guidelines suggest the adaption of treatment
duration based on a patients bacteriological
status or other markers for treatment
progress, there is no guidance concerning
the consequences of these markers, and the
meaning of other markers for treatment

treatment start
BIOMARKER-GUIDED
TREATMENT DURATION
INCREASING
DURATION OF
THERAPY
threshold

TREATMENT FAILURE

Pathogen-related Markers
Microscopy and Culture

TREATMENT REGIMEN

BIOMARKER LEVEL

FACTORS INFLUENCING DURATION OF TB THERAPY

disease severity, hosts immune status & genetic background,
bacterial virulence & drug-resistance, availability & quality of therapy

progress is not explained. Due to the

important inuence of host and pathogen
variables to achieve relapse-free cure with
currently available medications and the
overall long duration of anti-TB treatment
of M/XDR-TB, individualization of
the duration of M/XDR-TB treatment
would be highly desirable. In addition
to improvements in cost-effectiveness,
individualized treatment regimens achieve
higher success rates than standardized
treatment regimens (9) and may possibly
lead to better quality of life. However, the
only biomarker validated to guide clinicians
on decisions for the duration of therapy
in any bacterial infection to date is the
procalcitonin level in community-acquired
pneumonia (10).
The development of clinical and
radiological scores and identication of
host- and pathogen diseaserelated proles,
molecules, and signatures to monitor the
effect of anti-TB therapy is rapidly evolving.
The discovery of biomarkers to guide
clinicians for the duration of M/XDR-TB
treatment will have far-reaching clinical
and economic consequences.
This perspective elucidates the
expanding eld of TB biomarker research
for therapy response and introduces
potential future approaches for individually
tailored TB treatment durations, especially
needed for the management of M/XDR-TB.

pan drug-susceptible TB
TREATMENT PHASE

TB meningitis
rifampicin-resistant
TB

INTENSIVE
CONTINUATION

M/XDR-TB
0

12

18 20

DURATION OF TREATMENT (MONTHS)

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 of Mycobacterium
tuberculosis drug 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

In the early course of anti-TB treatment, the

most frequently used method to evaluate
a patients state of infectiousness is the
identication of acid-fast bacilli by sputum
smear microscopy (11). As a measure for
treatment 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 for
treatment outcome (1316) (Table 1).
In four different studies with patients
with MDR-TB under second-line drug
treatment, the median TCCs were reported
between 62 and 152 days (1317) compared
with representative 78 days in patients with
drug-susceptible TB (17). The median time
to smear conversion (SC) was 83 days in
patients with MDR-TB (16) and 23 days
in drug-susceptible TB (18), with similar
disease characteristics affecting TCC and
375

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

Marker
Pathogen-related markers
TCC
Whole blood bactericidal activity

TTD
TB-RNA
TB-DNA
TB-antigens
Host-related markers
Clinical items
Clinical score

Radiological parameters
Chest radiograph score
Chest CT scan

Classication

Time until a patient is considered

culture negative
Bactericidal activity against
Mycobacterium tuberculosis
in whole blood culture during
TB treatment
Time until a culture is positive
in days
Quantication of mycobacterial
RNA
Quantication of mycobacterial
DNA
Quantication of mycobacterial
antigens

Metabolomic patterns
Drug levels and drug activity
TB drug activity or
plasma concentration

Evaluated in
M/XDR-TB

Remarks

Sputum

Yes
No

Sputum

Yes

Sputum

No

MDR-TB with delayed TTD, treatment

response
Treatment response

Sputum, urine

Not specied

Treatment response

32, 35, 36, 38

Sputum, urine,
serum

Not specied

Treatment response

2430

Changes of patient
characteristics in the
course of treatment

Clinical score
items

Not specied

Treatment response

39, 40

Change of TB correlates
with treatment
TB correlates with TTD

Chest X-rays

Yes

4143

CT scans

Single drugresistant, no
M/XDR-TB
Not specied

Outcome prediction and treatment

response
Longer TTD in patients with
cavitary disease

18

F-FDG PET

Evaluated for survival prediction,

treatment response, and failure
Marker for treatment response

Reference

Whole blood

18
F-FDG PET
Decline of SUVmax
Chemokines, cytokines, proteins, and peptides
Acute-phase proteins/peptides
PCT, CRP, sTREM-1, heme
oxygenase-1, sICAM-1,
suPAR, sLAG-3,
granzyme B, sTNFR I,
and sTNFR II
Cytokines and
Among others IFN-g, TNF-a,
chemokines
IL-8, IL-6, IP-10, VEGF,
IL-10, MIP-1a, IL-13 and
sCD40L, MCP-1
Nutritional markers
Adiponectin, leptin, fetuin-A,
and retinol-binding protein
Release assays and specic cellular responses
Immunophenotype of specic
Different cytokine responses
cells
of antigen-specic T cells
and frequency changes
of these cells
Cell populations
Immune cells without specic
Expression of characterizing
stimulation
surface markers (i.e., CD25
and CD127 for Treg)
Antibodies
Circulating antibodies against
High throughput proof of TB
TB antigens
antigens (i.e., antibodies
against ESAT-6, CFP-10)
Proteomics and transcriptomics and gene expression
Gene-expression patterns
Different signatures for
distinct disease status
Single or small number gene
Candidate gene-expression
expression changes
changes
Protein patterns
Treatment response signatures

Mass spectrometry
VOC

Material

Treatment response

13, 16, 17
91

2023
31, 37

42

4447

Serum/plasma

No

Therapy response and therapy

outcome prediction

55, 92, 93

Sputum, serum,
plasma

No or not
specied

Therapy response and therapy

outcome prediction, correlation
with bacterial markers

51, 54, 94, 95

Plasma

Not specied

Severity of disease and treatment

response

96

Whole blood
and PBMC

Some studies

Therapy response, correlation with

pathogen-related markers and
outcome

63, 68, 71, 97

Whole blood
and PBMC

Some studies

Disease severity and treatment

response

Serum

No

Treatment response

99

Total RNA

No

Treatment response

83, 84, 86, 87

RNA

No

Treatment response

100

Serum
proteins

No

Treatment response

80, 81

Proof of mycobacterial
substances
Changes of metabolic
patterns

Breath

No

Treatment response

7678

Urine

No

Treatment response

75

Individual drug activity

Plasma

No

Different concentrations/activity predicts

outcome

71, 73, 98

101

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 proteins
1alpha; PCT = procalcitonin; PBMC = peripheral blood mononuclear cells; sCD40L = suluble cluster of differentiation 40 ligand; sLAG-3 = soluble
lymphocyte 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 standardized
uptake value; TB = tuberculosis; TCC = time to culture conversion; TNF = tumor-necrosis factor; TTD = time to detection; VEGF = vascular endothelial growth
factor; VOC = volatile organic compounds; XDR = extensively drug-resistant; 18F-FDG PET = fluorodeoxyglucose positron emission tomography.

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PULMONARY PERSPECTIVE
SC. However, culture- and smear-based
markers cannot serve as indicators for
positive therapy response after CC, and the
sensitivities for both 2-month smear (24%)
and culture (40%) results to predict relapse
were shown to be low (19). In the abovementioned studies, CC ranged from 62 to
152 days, representing a relatively short
duration of time in contrast to the entire
length of therapy and leading to a gap
of about 15 months between the last
positive culture and treatment end, as
recommended by the WHO. The decline
of colony-forming units on solid media
cultures or the time to culture positivity in
liquid media in the early phase of treatment
has been developed to assess the early
bactericidal activity of a single drug or
a combination of drugs (20, 21). Time
to culture positivity has already been
described as marker for treatment
response in patients with drug-susceptible
TB (22, 23). Possibly, analysis of serial
measurements of the time to culture
positivity during the early phase of
treatment in patients with M/XDR-TB
might help predict the total time needed for
individual relapse-free cure; however, at
this point in time this remains hypothetical.
Importantly, patients with MDR-TB not
achieving CC are more likely to fail therapy
(13), and reverting of cultures to positive
after CC or failure to achieve CC may be
a strong predictor for treatment failure.

Monitoring Antigens of
M. tuberculosis during Treatment

During infection, M. tuberculosisderived

antigens are released from the site of
infection. These are available for direct
identication in sputum, blood, urine, or
exhaled breath and have been evaluated
for diagnostic purposes (24, 25) and could
potentially be used also for monitoring
of TB treatment responses (2628) (Table
1). Limitations to this approach are low
levels of antigens available, necessitating
highly sensitive assays. At present,
mycobacterial lipoarabinomannan (LAM)
remains the only explored M. tuberculosis
antigen with sufcient specicity for
clinical use (24, 25). Concentrations of
LAM in urine specimens of patients with
active TB correlate with sputum bacillary
load (29). However, the sensitivity of the
LAM urine assays in patients with active
TB is too low to suggest that this target is
a promising marker to monitor treatment
Pulmonary Perspective

(24), especially in HIV-uninfected

patients (30).
To avoid delays that occur when
therapy response assessments depend
on M. tuberculosis growth in cultures,
quantication of M. tuberculosisspecic
nucleic acids by amplication methods are
a more rapid alternative for treatment
monitoring. Results can be available within
hours. Whereas M. tuberculosisspecic
DNA can remain detectable also long
after sterile cure, the identication of
M. tuberculosisspecic RNA products,
which are shorter lived, is a promising
method to identify viable bacteria (31).
Another interesting approach includes
treating samples with propidium monoazide,
which can bind the DNA from nonviable
bacteria and prevent it from being amplied
by polymerase chain reaction; thus only
viable organisms are detected (32).
Monitoring Nucleic Acids of
M. tuberculosis during Treatment

In principle, M. tuberculosisspecic
nucleic acids can be isolated from different
compartments, such as sputum and urine
(33, 34). However, in a recent direct
comparison quantication of sputum
M. tuberculosis, DNA was inferior compared
with culture-guided methods to assess TB
treatment responses (35). The reduction of
detectable M. tuberculosis DNA measured
by the Xpert MTB/RIF was too poor in
specicity to serve as reliable marker for
treatment response monitoring (36). In
contrast, RNA is a marker for viable
bacteria, and the decline of quantiable
mycobacterial RNA in sputum specimen
of patients with TB under treatment has
recently been introduced as promising
marker to monitor early treatment
responses in an early bactericidal activity
study (37) (Table 1). Monitoring the
excretion of M. tuberculosisspecic nucleic
acids in the urine of patients with TB after
treatment initiation could serve as a marker
for bacterial killing (38) and may have
advantages over sputum analyses for
treatment monitoring.
With currently available technologies,
all pathogen-related markers for TB therapy
responses have a short detection phase in
relation to the total recommended duration
of anti-TB treatment. Pathogen-related
markers might be better suitable to identify
treatment failures and relapses (31, 37)
rather than to serve as markers of successful
treatment outcome or the time for

treatment duration. As the risk for relapse

is substantially increased when treatment
is discontinued at the time when bacteria
are not directly or indirectly identied by
currently available methods, host-related
markers could be more appropriate to
identify the individual duration of anti-TB
therapy.

Host Markers
Clinical Scores

A simple way to evaluate patient response to

treatment is the observation of changes in
general clinical characteristics of patients
with TB. Low-cost clinical scoring systems
have been developed to predict mortality
and treatment failure and to monitor
treatment response (39, 40). The value
of such scores depends on baseline
characteristics of the patients with TB,
the clinical setting, and the training,
commitment, and accuracy of the medical
staff recording the data. In contrast to
patients with minimal disease, people with
advanced stages of the disease should have
the highest probability for changes in
the clinical score on positive treatment
responses. Although studies are ongoing to
compare clinical scores in patients with
different levels of drug-resistant TB, it is
speculative at this moment whether slower
bacterial clearance in M/XDR-TB is also
related to the kinetic of clinical scores
before and after culture conversion,
especially in the continuation phase
of the treatment.
Clinical scores, although appealing due
to their simplicity and low cost, are likely not
sensitive enough to indicate the end of
treatment and are subject to a high degree
of inter- and intraobserver variability.
However, it should be explored whether
a combination of a clinical score with
bacterial/host-derived markers or results
of imaging studies may be able to serve
as instruments for therapy monitoring.
Imaging

Conventional chest X-rays and computed

tomography (CT) scans are used to assess
the extent of disease before treatment
initiation and at intervals in the course of
treatment (41, 42) (Table 1). A simple
numerical score describing the extent of
pathological changes on conventional chest
radiographs has been suggested by Ralph and
colleagues (41). However, radiological
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PULMONARY PERSPECTIVE
scoring systems may not be useful to
monitor therapy-associated changes in
patients with MDR-TB. A recent study with
South African patients with MDR-TB could
not show a signicant correlation of
consolidations or cavitation in baseline
radiographs with time to culture positivity
(43). A highly sensitive imaging method
that is able to show viable mycobacteria
would be ideal to determine the duration of
anti-TB therapy. CT scans are a candidate
as treatment-monitoring tools because the
initial number of cavities in CT scans has
been shown to correlate with increase
of time to positivity in liquid culture
under treatment (42). Alternatively, 18Fuorodeoxyglucose positron emission
tomography (FDG-PET) CT has been
explored as a method to monitor treatment
responses in TB in animal models as well as
in humans (4447). 18F-FDG PET CT was
directly compared with microbiological
methods and was able to identify
bactericidal activity of drug regimens in
mice (45). Findings in a rabbit model
characterized the behavior of TB lesions
under treatment with rifampicin or
isoniazid (46). 18F-FDG PET CT was able
to indicate for therapy response by the
decline of maximum standardized uptake
values of TB lesions (44). Still, the tracers
uptake properties in scar tissue in contrast
to active TB lesions have to be elucidated to
further characterize the diseases behavior
and to derive specic signs for treatment
response. As an alternative to 18F-FDG PET
CTmediated imaging, other radiotracers
could indicate ongoing inammatory sites
of infection but are not applicable for
large trials yet (48, 49). Costs and lack of
accessibility obviously limit the use of these
sophisticated techniques in high-burden
countries.
Immunological Markers
Cytokines and proteins. Inammatory states
of patients with active disease can be
monitored by analyses of peptides and
cytokines in blood specimens during
treatment (Table 1). Treatment initiation
results in a rapid killing of susceptible
mycobacteria and increase in the release
of M. tuberculosis antigens (50). This will
transiently augment the activity in the
already primed adaptive immune system,
resulting in increased cell-mediated
immune activity, again driving release of
nonspecic mediators such as acute-phase

378

reactants. Providing that these signatures

normalize in the course of treatment, the
kinetics of such markers may indicate
therapy response and could help clinicians
to better evaluate and assess individual
treatment in patients with M/XDR-TB
(Figure 1). Recently, plasma specimens
of 42 patients with TB were investigated
weekly over a total period of 18 months
(51). Among 24 measured cytokines,
IFN-ginducible protein 10 and vascular
endothelial growth factor levels were the
only candidate markers that showed
signicant changes in the course of
treatment in HIV-positive and -negative
patients. In fact, vascular endothelial
growth factor levels measured after 2 weeks
of therapy correlated with the time of
sputum smear conversion. These ndings
require validation in larger prospective
cohorts and especially for patients with
M/XDR-TB. There are numerous protein
biomarker candidates described in the
literature with different potential for
therapy response as surrogate endpoint
(5255). Up to now, no single marker or
panel of markers is recommended for
clinical routine use as a biomarker to guide
the duration of TB therapy. Some markers
have already been explored in patients with
immunosuppression (i.e., HIV infection)
(51), but the hampered immune responses
of these patients could limit the use of
these markers. Still, protein markers are
methodically attractive because point-of-care
tests can be easily developed (i.e., lateral
ow-test platforms) and specimens are easy
to obtain (i.e., capillary blood).
Antigen-specic immune responses
and immune-cell phenotypes. The most

accurately described methods for identifying

M. tuberculosisspecic immune responses
are the tuberculin skin test and IFN-g
release assay (IGRA). Presence of a positive
tuberculin skin test or IGRA result denes
latent infection with M. tuberculosis
in the absence of active TB (56). In
routine clinical practice, ELISA IGRAs
(QuantiFERON-TB Gold In-Tube test;
Qiagen, Hilden, Germany) or ELISPOT
IGRAs (T-Spot.TB test; Oxford
Immunotec, Abingdon, UK) are used to
evaluate sensitization to M. tuberculosis
antigens (early secretory antigenic target 6
[ESAT-6] and 10-kD culture ltrate antigen
[CFP-10]), which is indicated by the release
of IFN-g of specic effector T cells (57, 58).
Prospective evaluations of IGRA changes
over time have not shown promise for use

as monitoring tools in active TB; IGRAs

show substantial intraindividual variation
and do not correlate with clinical endpoints
such as smear or culture conversion (59).
Identication of up-regulation of
stress-specic antigens caused by effective
TB treatment could be an attractive method
to identify treatment responses in TB.
The IFN-g response of antigen-specic
T cells from patients with TB and
household contacts to different phasespecic antigens, including 24 antigens
of starvation and stress phases of
M. tuberculosis, were analyzed (60). There
was no difference of IFN-g responses
comparing patients with active TB and
household contacts. Cellular responses of
1,247 patients with TB to 23 disease-specic
antigens revealed substantial heterogeneity
of IFN-g responses due to different
disease states, HIV status, and geographical
background (61). Using disease stagespecic
targets may improve cytokine release
assays as tools to monitor M/XDR-TB
treatment responses (62).
Recently, IFN-g production by
M. tuberculosisspecic CD81 T lymphocytes
has been found to decrease signicantly
during the rst 6 months of TB treatment
(63). Still, compared with other cytokines
that are released by antigen-specic cells in
response to infection with M. tuberculosis,
IFN-g alone may not be the best cytokine
marker to evaluate therapy responses in
TB (64). For the discrimination between
active TB and latent TB infection (LTBI),
secretion of TNF-a by antigen-specic
CD4 T cells detected using intracellular
cytokine-staining uorescence-activated
cell sorting (FACS-ICS) was shown to
be superior to IFN-g (65). One study
suggested that the frequency of single TNFa1CD41 T cells with effector memory
phenotype (CD45RA2CCR72CD1272)
can discriminate between active TB and
LTBI with a very high sensitivity of up to
99% (66). IL-2 is another cytokine that has
been explored as a marker for treatment
responses in TB (67, 68). During the
course of TB treatment, a shift from
predominantly IFN-gproducing antigenspecic T cells toward predominantly IL2producing antigen-specic T cells can be
observed, suggestive of shift from effector
to central-memory T-cell dominance and
infection control (68). Along these lines,
it was shown that a low frequency of
IFN-g/IL-2 positive CD41 T cells in
response to puried protein derivative

American Journal of Respiratory and Critical Care Medicine Volume 190 Number 4 | August 15 2014

PULMONARY PERSPECTIVE
has been shown indicative for active TB
(67). Although data are still limited,
continuous follow-up of patients for
FACS of antigen-specic T cells appears
to be a promising method to monitor
treatment responses and warrants further
exploration. FACS methods are able to
characterize more complex and detailed
analysis of immune cell phenotypes
compared with IGRAs. However, as only
few M. tuberculosisspecic T cells are
available for interrogation from whole
blood, the FACS methods remain difcult
to standardize and perform with adequate
reproducibility (69). Fluorescenceimmunospot is an emerging simpler
technology that allows for two-cytokine
read-out of antigen-specic T cells. This
assay has shown promise as marker for
pathogen burden (70).
Rare immune-cell populations
(i.e., gdT cells, mucosa-associated invariant
T cells, and exhaustion T cells) seem
attractive for treatment monitoring of
M/XDR-TB (7173). Programmed-death-1
(PD-1, CD279) is a cell-surface receptor
of the immunoglobulin superfamily found
on immune effector cells and is a marker
on exhaustion T cells, which play in chronic
inammatory states. PD-11 and its ligands
are expressed on T cells, monocytes, and
B cells in patients with active TB, and
expression on these cells declines during TB
therapy (71). The ex vivo blockade of PD-1
rescues M. tuberculosisspecic IFNgproducing T cells from undergoing
apoptosis (71).
Circulating regulatory T cells (Treg,
CD41CD25high1CD1272FoxP31) have
been evaluated before and 6 months
after the resection of cavitary pulmonary
lesions in patients with MDR-TB (74).
Compared with healthy control subjects,
there were higher frequencies of Tregs
before surgery with a signicant decline
to levels, which were comparable to
healthy control subjects at 6 months
after surgery.
X-Omics

High-throughput analysis of metabolites,

proteins, or transcription products
from blood products, urine, or sputum
can provide disease stagespecic
molecular patterns, so-called -omics
(i.e., metabolomics, proteinomics,
transcriptomics, or mass spectrometry)
to unravel previously unidentied
biosignatures.
Pulmonary Perspective

Metabolomics analysis using mass

spectrometry was recently applied to
urine samples from patients undergoing
treatment for TB (75). A 23 small molecular
metabolitebased TB early treatment
response biosignature was shown to
consistently change in abundance between
baseline and across the treatment period
(75). This signature strongly differentiates
treatment effect early, suggesting a clinically
useful measure for treatment effect from an
attractive sample.
Volatile organic compounds (VOCs) in
exhaled breath specimens are currently
being explored as screening techniques for
active pulmonary TB (76). Comparable to
an alcohol breath test, minute amounts
of aromatic compounds and fatty-acid
derivatives (i.e., camphor, methyl
butenolide phenol, and methyl dimethyl
benzoate), specic for M. tuberculosis, can
be traced by analytic detectors (77, 78).
VOC detection technologies have not been
developed beyond the stage of prototypes
and not been validated to monitor the
course of TB treatment. At present it seems
unlikely that VOCs will be present in large
enough amounts in the exhaled breath
after culture conversion to determine
the end of TB treatment accurately (79).
Although the complexity of the current
available detection methods restricts eld
applicability, the identication of VOC,
urine, or plasma biosignatures has shown
promise for next-generation TB diagnostics
and treatment monitoring (Table 1).
Aptamer proteomic approaches
discovered serum signatures related to host
defense, acute-phase response, and pattern
recognition mechanisms in patients on
treatment for drug-susceptible TB (80, 81).
Expression levels of acute-phase proteins
(e.g., C-reactive protein) changed most
prominently during the rst 8 weeks of
anti-TB treatment (81). Using a similar
approach, the 8-week M. tuberculosis
culture status could be predicted by
a treatment response biosignature
consisting of ve serum proteins involved
in the coagulation cascade, neutrophil
activity, immunity, inammation, and
tissue remodelling with a sensitivity of 95%
and a specicity of 90%, respectively (80).
For transcriptomics approaches, RNA
arrays methods are capable to assess upand down-regulation of total RNA of all
circulating peripheral blood cells. In a
large African cohort, active TB could be
differentiated from LTBI in HIV-positive

and -negative patients by RNA expression

proles (82). A neutrophilic-driven, IFNginducible 393-transcript signature
specic for active TB, which could be
correlated with radiological extent, has been
revealed by a large-scale total-RNA array
method (83). Besides mainly IFN-gdriven
signatures, there were Fc-Gamma receptor
and type-I IFN networks discovered to
be the main responsive systems in active
TB (84, 85). Importantly, changes in
the gene-expression proles could be
demonstrated after the initiation of TB
therapy (86, 87). In these studies (83, 86),
the individual changes and the transcript
signature after treatment were very much
comparable to the ones of healthy control
subjects or patients with LTBI. Signature
expressions showed very little change
between 6 months (therapy end) and
follow-up after 12 months in patients with
pandrug-susceptible TB, indicating for
a robust cure signature for the time of
follow-up. Although gene expression
signatures are promising tools to monitor
treatment responses in TB, they have not
yet been evaluated for this purpose.

Conclusions
Although substantial advances have been
made to characterize the path from disease
to cure in individual patients, we still
use standardized treatment regimen and
durations for the therapy of patients with
mycobacterial infections. Recently there is
an emerging interest to explore biomarkers
and biosignatures that could guide clinicians
to determine the duration of TB therapy
for individual patients. Candidate markers
are still in preclinical or early clinical
evaluation, and none of these markers
can be recommended at present for use
in routine clinical practice. Such markers
are urgently needed, especially for the
management of patients with M/XDR-TB,
where treatment responses are highly
variable and current recommendations for
treatment durations may not be adequate
for the majority of affected individuals (88).
At present, treatment against M/XDR-TB
is characterized by a high frequency of
adverse events, high costs, and a poor
prognosis. Individualization of the duration
of M/XDR-TB treatment, rather than
standardized therapy, could become of
global relevance for the management of
M/XDR-TB if it improves quality of life,
379

PULMONARY PERSPECTIVE
proves to be cost effective, and ensures
a better treatment outcome (88).
In contrast to clinical trials, where
different stages of evaluation provide
a possible clue when new drugs may become
available for clinical use, the development
process for new diagnostic methods is less
predictable. However, the resurrected
interest in TB drug trials provides excellent
opportunities to link therapeutic and

diagnostic evaluations and to study

biomarkers and biosignatures that could
identify the time-point of relapse-free cure
independent of comorbidities, immune
status, and pathogen virulence (89, 90). The
validation process of such markers in large
prospective cohorts with biomarker-guided
therapy duration implies long-term close
observation follow-up periods. Still,
individualizing the duration of TB therapy

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