New approaches in the diagnosis and treatment of latent tuberculosis infection
Ahmad Respiratory Research
New approaches in the diagnosis and treatment of latent tuberculosis infection
0 Department of Microbiology, Faculty of Medicine, Kuwait University , Kuwait
With nearly 9 million new active disease cases and 2 million deaths occurring worldwide every year, tuberculosis continues to remain a major public health problem. Exposure to Mycobacterium tuberculosis leads to active disease in only ~10% people. An effective immune response in remaining individuals stops M. tuberculosis multiplication. However, the pathogen is completely eradicated in ~10% people while others only succeed in containment of infection as some bacilli escape killing and remain in non-replicating (dormant) state (latent tuberculosis infection) in old lesions. The dormant bacilli can resuscitate and cause active disease if a disruption of immune response occurs. Nearly one-third of world population is latently infected with M. tuberculosis and 5%-10% of infected individuals will develop active disease during their life time. However, the risk of developing active disease is greatly increased (5%-15% every year and ~50% over lifetime) by human immunodeficiency virus-coinfection. While active transmission is a significant contributor of active disease cases in high tuberculosis burden countries, most active disease cases in low tuberculosis incidence countries arise from this pool of latently infected individuals. A positive tuberculin skin test or a more recent and specific interferon-gamma release assay in a person without overt signs of active disease indicates latent tuberculosis infection. Two commercial interferon-gamma release assays, QFT-G-IT and T-SPOT.TB have been developed. The standard treatment for latent tuberculosis infection is daily therapy with isoniazid for nine months. Other options include therapy with rifampicin for 4 months or isoniazid + rifampicin for 3 months or rifampicin + pyrazinamide for 2 months or isoniazid + rifapentine for 3 months. Identification of latently infected individuals and their treatment has lowered tuberculosis incidence in rich, advanced countries. Similar approaches also hold great promise for other countries with low-intermediate rates of tuberculosis incidence.
Tuberculosis (TB) is a formidable public health
challenge as it contributes considerably to illness and death
around the world. The most common causative agent of
TB in humans, Mycobacterium tuberculosis, is a member
of the M. tuberculosis complex (MTBC) which includes
six other closely related species: M. bovis, M. africanum,
M. microti, M. pinnipedii, M. caprae and M. canettii.
All MTBC members are obligate pathogens and cause
TB; however, they exhibit distinct phenotypic properties
and host range. Genetically, MTBC members are closely
related, the genome of M. tuberculosis shows >99.9%
similarity with M. bovis, the species that primarily
infects cattle but can also cause TB in other mammals
including man [1,2]. The current TB epidemic is being
sustained by two important factors; the human
immunodeficiency virus (HIV) infection and its association
with active TB disease and increasing resistance of M.
tuberculosis strains to the most effective (first-line)
antiTB drugs [3-5]. Other contributing factors include
population expansion, poor case detection and cure
rates in impoverished countries, wars, famine, diabetes
mellitus and social decay and homelessness [6,7].
According to recent estimates, 9.4 million new active
disease cases corresponding to an estimated incidence of
139 per 100,000 population occurred throughout the
world in 2008 [3,4]. Only 5.7 million of 9.4 million cases
of TB (new cases and relapse cases) were notified to
national tuberculosis programs of various countries
while the rest were based on assessments of
effectiveness of surveillance systems. The highest number of TB
cases occurred in Asia (55%) followed by Africa (30%).
The highest incidence rate (351 per 100,000 population)
was recorded for the African region, mainly due to high
prevalence of HIV infection. An estimated 1.4 million
(15%) of incident TB patients were coinfected with HIV
in 2008. Globally, the total prevalent TB cases in 2008
were 11.1 million corresponding to 164 cases per 100
000 population that resulted in 1.8 million deaths
(including 0.5 million TB patients coinfected with HIV)
[3,4]. Nearly 440 000 cases of multidrug-resistant TB
(MDR-TB, defined as infection with M. tuberculosis
strains resistant at least to the two most important
firstline drugs, rifampicin and isoniazid) occurred in 2008
. By 2009, extensively drug-resistant TB (XDR-TB;
defined as MDR-TB strains additionally resistant to a
fluoroquinolone and a second-line anti-TB injectable
agent such as kanamycin, amikacin, or capreomycin) has
been found in 58 countries . While MDR-TB is
difficult and expensive to treat, XDR-TB is virtually an
untreatable disease in most of the developing countries
Establishment and persistence of latent M.
Tuberculosis is a communicable disease and infection is
initiated by inhalation of droplet nuclei (1-5 μm in
diameter particles) containing M. tuberculosis, expectorated
by patients with active pulmonary or laryngeal TB,
typically when the patient coughs. Active transmission
occurs more frequently in small households and
crowded places in countries with a high incidence of TB
and the risk of infection is dependant on several factors
such as the infectiousness of the source case, the
closeness of contact, the bacillary load inhaled and the host’s
immune status (Figure 1) [9-11]. Molecular
epidemiological studies have shown that there are distinct
differences in the disease presentation and population
demographics in low TB incidence and high TB
incidence countries. In several African and Asian countries,
the vast majority of mycobacterial infections are caused
by M. tuberculosis and incidence rates are highest
among young adults, with most cases resulting from
recent episodes of infection or reinfection [12-14]. On
the contrary, in low TB incidence countries of Western
Europe and North America, a higher proportion of
active TB cases occur in older patients or among
immigrants from high TB incidence countries .
Pulmonary TB accounts for >85% of active TB cases in high TB
incidence countries while extrapulmonary TB is more
common in low TB incidence countries, particularly
among HIV infected individuals and immigrants
originating from TB endemic countries [15,16].
The inhaled droplet nuclei avoid the defenses of the
bronchi due to their small size and penetrate into the
terminal alveoli of the lungs where they are engulfed by
phagocytic antigen-presenting cells including alveolar
macrophages, lung macrophages and dendritic cells. In
the lungs, M. tuberculosis can also infect non-phagocytic
cells in the alveolar space such as endothelial cells, M
cells and type 1 and type 2 epithelial cells [17-20]. In
the initial phase of infection, M. tuberculosis internalized
by macrophages and dendritic cells replicates
intracellularly and the bacteria-laden immune cells may cross the
alveolar barrier to cause systemic dissemination [18,19].
The intracellular replication and simultaneous
dissemination of the pathogen to the pulmonary lymph nodes
and to various other extrapulmonary sites occurs prior
to the development of the adaptive immune responses
The entry of M. tuberculosis in phagocytic immune
cells in the alveolar space begins with recognition of
pathogen-associated molecular patterns by specific
pathogen recognition receptors that initiate a
coordinated innate immune response by the host . The M.
tuberculosis components are recognized by host
receptors that include toll-like receptors (TLRs),
nucleotidebinding oligomerization domain (NOD)-like receptors
(NLRs), and C-type lectins [24-26]. The C-type lectins
include mannose receptor (MR), the dendritic
cell-specific intercellular adhesion molecule grabbing nonintegrin
(DC-SIGN), macrophage inducible C-type lectin
(Mincle) and dendritic cell-associated C-type lectin-1
(Dectin-1) [24,27]. The TLR signaling is the main arm of the
innate immune response and M. tuberculosis
internalized through different receptors may also have different
The M. tuberculosis cell envelope is composed of a
cell wall that is covered with a thick waxy mixture of
lipids, polysaccharides and mycolic acids. The most
important M. tuberculosis cell surface ligands that
interact with TLRs and other receptors include the 19 and
27 kDa lipoproteins, 38 kDa glycolipoprotein, glycolipids
(such as phosphatidylinositol mannoside, PIM;
lipomannan, LM; lipoarabinomannan, LAM; and
mannosecapped lipoarabinomannan, Man-LAM) and trehalose
dimycolate (TDM) (Table 1) [26,28,30,31]. Other ligands
may include surface exposed proteins such as LprA and
LprG lipoproteins and mammalian cell entry (Mce)
proteins encoded by the mce1 and mce3 operons [32-36].
Typically, signals generated through TLR and Mincle
promote proinflammatory immune responses while
preferential recruitment of DC-SIGN induces suppression
and/or exhaustion of immune responses [25,27,30,37].
The glycolipids (such as PIM, LM and, LAM) and
lipoproteins (such as 19 kDa lipoprotein, LpqH) that are
exposed on M. tuberculosis cell surface  are mainly
recognized by TLR2 (Table 1) [24,26,30].
The interaction of M. tuberculosis ligand(s) with TLRs
initiates an intracellular signaling cascade that
culminates in a proinflammatory response (beneficial to the
Figure 1 Natural progression of events and outcome in an immunocompetent individual following exposure of human subjects
(contacts of TB patients) to droplet nuclei containing M. tuberculosis expectorated by a source case of sputum smear-positive
pulmonary TB. Every year, ~50 million people worldwide are infected with M. tuberculosis. Complete elimination of tubercle bacilli is achieved in
~10% individuals only while in ~90% of infected individuals, bacterial growth is stopped but some bacilli survive and persist leading to latent M.
tuberculosis infection (LTBI). The waning of dormant bacilli in persons with LTBI can be accelerated by therapy with isoniazid for 9 months
(denoted by *). The vaccines currently in clinical trials are designed to prevent or delay the reactivation of latent infection in persons with LTBI
(denoted by **).
Table 1 Important M. tuberculosis ligands, main receptors on phagocytic immune cells and immune cell processes
affected that promote persistence of the pathogen and establishment of latent tuberculosis infection in humans
Phosphatidyinositol mannoside (PIM)
M. tuberculosis liganda
19 kDa Lipoprotein (LpqH)
19 kDa Lipoprotein (LpqH)
Trehalose dimycolate (cord factor)
Trehalose dimycolate (cord factor)
Host cell receptorb
Immune cell process affected
MHC class II expression/antigen presentation
Phagosomal processing by MHC class I pathway
MHC class II expression/antigen presentation
MHC class II expression/antigen presentation
Modulation of macrophage signaling pathways
Modulation of macrophage signaling pathways
Modulation of macrophage signaling pathways
MHC class II expression/antigen presentation
IL-12 secretion of dentritic cells/macrophages
Apoptosis of macrophages
MHC class II expression/antigen presentation
aMannose-capped LAM, Mannose-capped lipoarabinomannan
bTLR2, Toll-like receptor 2; MR, mannose receptor; DC-SIGN, dendritic cell-specific intercellular adhesion molecule grabbing nonintegrin; Mincle, macrophage
inducible C-type lectin
host), however, the bacterium has also evolved strategies
that can trigger signals that dampen the innate immune
response (beneficial to the pathogen). The
proinflammatory process results in activation of nuclear transcription
factor (NF)- B and production of proinflammatory
cytokines, chemokines and nitric oxide through either
myeloid differentiation primary response protein 88
(MyD88)-dependant or MyD88-independent pathway
[24,30,39-41]. A brief outline of the immune response of
the host is described here. Several excellent review
articles are available for a more detailed description
In addition to macrophages and dendritic cells, a wide
range of other immune components are also involved in
an effective immune response against M. tuberculosis
and include, ab-T cells (both CD4+ and CD8+), CD1
restricted T cells, gδ-T cells and cytotoxic T cells as well
as the cytokines produced by these immune cells
[25,45-47]. The most important among these are CD4+
T cells and the cytokine interferon (IFN)-g.
The two major defense mechanisms of macrophages
include the fusion of the phagosomes containing M.
tuberculosis with lysosomes (phagolysosome) that is
bactericidal and generation of nitric oxide and other
reactive nitrogen intermediates (RNI) which exert toxic
effects on the bacilli [43,45,48-51]. The M. tuberculosis
containing phagosomes mature through a series of
fusion and fission events with several endocytic vesicles
that culminate in a phagolysosome. The fusion-fission
events remodel the phagosomal membrane. The Ca+2
signaling cascade and recruitment of vacuolar-proton
transporting ATPase (vH+-ATPase) cause lowering of
internal pH that allows lysosome-derived acid hydrolases
to function efficiently for their microbicidal effect
[52-54]. Another mycobactericidal mechanism of
macrophages includes lysosomal killing of M. tuberculosis
mediated by ubiquitin-derived peptides . The
ubiquitination destroys tubercle bacilli by autophagy as a
ubiquitin-derived peptide impairs the membrane integrity
of M. tuberculosis that allows nitric oxide to kill more
efficiently. The apoptosis of infected macrophages
participates in host defense against infection as apoptotic
vesicles containing mycobacterial antigens are taken up
by dendritic cells for CD8+ T cell activation by
phagosome-enclosed antigens [25,56,57].
Mycobacterial antigens in macrophages or dendritic
cells are picked up by the MHC class II molecules and
presented to CD4+ T cells [28,32,43]. The phagosomal
membrane is also equipped with the MHC class I
processing machinery [58,59]. Also, CD1 proteins present
glycolipids, lipids, and lipopeptides of lipid-rich M.
tuberculosis to T cells [56,60,61]. Furthermore, the
vesicles formed due to apoptosis of M. tuberculosis-infected
macrophages are taken up by dendritic cells and
presented to the T cells through the MHC class I and CD1
Immediately after entry of M. tuberculosis, alveolar
macrophages produce inflammatory cytokines and
chemokines that serve as a signal for infection. The
monocytes, neutrophils and lymphocytes migrate to the focal
site of infection but they are unable to kill the bacteria
efficiently. During this time, the bacilli resist the
bactericidal mechanisms of the macrophage (phagolysosome)
by preventing phagosome-lysosome fusion, multiply in
the phagosome and eventually escape from phagosome/
phagolysosome and cause macrophage necrosis [44,51].
The escape of M. tuberculosis from
phagosome/phagolysosome is aided by the 6-kDa early secreted antigenic
target (ESAT-6) protein and ESX-1 protein secretion
system encoded by the region of difference 1 (RD1), a
genomic segment that is present in all virulent M.
tuberculosis and M. bovis strains but is absent in the vaccine
strain M. bovis BCG [1,2,62-68]. The ESAT-6 protein
associates with liposomes containing
dimyristoylphosphatidylcholine and cholesterol and causes
destabilization and lysis of liposomes . It has also been shown
that ESAT-6, released during acidification of phagosome
from ESAT-6:10 kDa-culture filtrate protein (CFP-10)
complex (secreted by live M. tuberculosis through ESX-1
secretion system), inserts itself into lipid bilayer and
causes lysis of phagosome and escape of tubercle bacilli
. The ESAT-6 also induces apoptosis of
macrophages via the caspase-dependent pathway and cytolysis
of type 1 and type 2 alveolar epithelial cells and helps in
the dissemination of M. tuberculosis [20,70].
The released bacilli multiply extracellularly, are
phagocytosed by another macrophage that also fails to control
the growth of M. tuberculosis and likewise is destroyed
[42,43,51,71,72]. This progression of events continues
unabated (in persons with a weak immune response)
leading to active TB disease in ~10% of individuals
(Primary TB) (Figure 1). In vast majority of the infected
individuals, however, an effective cell-mediated immune
response develops 2-8 weeks after infection as dendritic
cells with engulfed bacilli mature, migrate to the
regional lymph node and prime T cells (both CD4+ and
CD8+) against M. tuberculosis antigens [25,45,73]. The
specific immune response produces primed T cells
which migrate back to the focus of infection, guided by
the chemokines produced by infected cells. The
accumulation of macrophages, T cells and other host cells
(dendritic cells, fibroblasts, endothelial cells and stromal
cells) leads to the formation of granuloma at the site of
infection [74,75]. The CD4+ T cells producing IFN-g
recognize infected macrophages presenting antigens
from M. tuberculosis and kill them [43,45,76].
The early stages of granuloma formation appear to
benefit M. tuberculosis as ESAT-6 promotes
accumulation of macrophages of different activation and
maturation stages at the site of infection in which the tubercle
bacilli multiply unabated and infected macrophages may
also transport the pathogen to other sites in the body
[22,77]. The eventual formation of solid granuloma due
to an effective immune response walls off tubercle bacilli
from the rest of the lung tissue, limits bacterial spread
and provide microenvironment for interactions among
macrophages and other immune cells and the cytokines.
It is also apparent now that M. tuberculosis infected
individuals show differences in the innate immune
responses that lead to the formation of physiologically
distinct granulomatous lesions. Some of these lesions
eliminate all bacilli (sterilizing immunity) while others
allow persistence of viable M. tuberculosis in the
microenvironment [75,78]. Low-dose infection in primate
models of human latent TB exhibit at least two types of
tuberculous granuloma [79,80]. The classic caseous
granuloma are composed of epithelial macrophages,
neutrophils, and other immune cells surrounded by
fibroblasts. M. tuberculosis resides inside macrophages
in the central caseous necrotic region that is hypoxic
[80,81]. The second type of granulomas (fibrotic lesions)
are composed of mainly fibroblasts and contain very few
macrophages, however, the exact location of viable M.
tuberculosis in these lesions is not known .
With granuloma formation and an effective immune
response, most tubercle bacilli are killed and disease
progression is halted [42,45,75]. Although
proinflammatory immune response is generally beneficial to the host,
restricting this response is essential to avoid the risk of
producing excessive inflammation that could damage
host tissues. This is accomplished through a family of
receptor tyrosine kinases that provide a negative
feedback mechanism to both, TLR-mediated and
cytokinedriven proinflammatory immune responses [82,83]. This
defense mechanism of the host has been exploited by
M. tuberculosis for its survival [84-87]. Several M.
tuberculosis factors such as 19-kDa lipoprotein, glycolipids
(particularly Man-LAM), trehalose dimycolate (cord
factor) and several others (Table 1) can modulate
antigenprocessing pathways by MHC class I, MHC class II and
CD1 molecules, phagolysosome biogenesis and other
macrophage signaling pathways [26-28,30,32,33,88-95].
The suppression of these responses blunt the
microbicidal functions of macrophages and other immune
cells (such as reactive nitrogen intermediates) or
prevent their proper maturation (phagolysosome)
The inhibition of macrophage responses to M.
tuberculosis results in a subset of infected macrophages that
are unable to present M. tuberculosis antigens to CD4+
T cells. This results in insufficient activation of effector
T cells leading to evasion of immune surveillance and
creation of niches where M. tuberculosis survives
[45,51,96,97]. The hypoxia, nutrient deficiency, low pH
and inhibition of respiration by nitric oxide in the
microenvironment of the granuloma induce a dormancy
program in M. tuberculosis [98,99]. These conditions
transform surviving bacilli into a dormant stage with
little or no metabolic and replicative activity, however,
expression of DosR-regulated dormancy antigens
continues [99-101]. It is also probable that M. tuberculosis,
under these conditions, forms spore-like structures,
typically seen with other mycobacteria in response to
prolonged stationary phase or nutrient starvation, for its
survival . Decreased outer membrane permeability
also protects M. tuberculosis from killing by
ubiquitinderived peptides . Thus, some non-replicating
(resistant) bacilli avoid elimination by the immune
system and persist. This latent tuberculosis infection
(LTBI) in a person without overt signs of the disease is
indicated by the delayed-type hypersensitivity (DTH)
response to purified protein derivative (PPD) prepared
from culture filtrates of M. tuberculosis (tuberculin skin
test) [9,104]. The dormant bacilli can inhabit the
granuloma during the lifetime of the host but are able to
resume their growth if (or when) the immune response
is compromised (reactivation TB) (Figure 1). The World
Health Organization (WHO) has estimated that
onethird of the total world population is latently infected
with M. tuberculosis and 5%-10% of the infected
individuals will develop active TB disease during their life
time . However, the risk of developing active
disease is 5%-15% every year and lifetime risk is ~50% in
HIV coinfected individuals [3,4,105].
Reactivation of latent infection requires M.
tuberculosis to exit dormancy. The lytic transglycosylases known
as resuscitation promoting factors and an endopeptidase
(RipA) of M. tuberculosis have been recognized as vital
components for revival from latency [106-108].
Although reactivation of latent infection can occur even
decades after initial infection, a person is at greater risk
of developing active TB disease during the first two
years after infection with M. tuberculosis [9,109,110].
Several factors can trigger development of active disease
from reactivation of remote infection, and typically
involve the weakening of the immune system . HIV
infection is the most important risk factor for
progression to active disease in adults as it causes depletion/
functional abnormalities of CD4+ and/or CD8+ T-cells
that are central for protection against active TB disease
[3,4,6,105]. Likewise, M. tuberculosis infection
accelerates the progression of asymptomatic HIV infection to
acquired immunodeficiency syndrome (AIDS) and
eventually to death. This is recognized in the current AIDS
case definition as pulmonary or extrapulmonary TB in
HIV-infected patient is sufficient for the diagnosis of
AIDS. The reactivation TB can occur in any organ
system, however, in immunocompetent individuals, it
usually occurs in the upper lobes, where higher oxygen
pressure supports good bacillary growth.
New dynamic model of latent tuberculosis
The traditional model of LTBI as described in detail
above begins with the entry of M. tuberculosis in
antigen-presenting cells in lung alveoli and the pathogen
accomplishes intracellular survival through several
evasion strategies including neutralization of the
phagosomal pH, antigen presentation by macrophages and
dendritic cells that compromise CD4+ T cell stimulation,
apoptosis of infected macrophages and interference with
autophagy [51,75,111,112]. The early stages of
developing granuloma benefit the pathogen as it invades
macrophages of different activation and maturation stages and
thus, survives when the loose aggregates of phagocytes
and polymorphonuclear granulocytes transform into a
solid granuloma [75,77,111]. Although active disease is
averted for the moment, latent infection ensues as the
pathogen is not eliminated. The tubercle bacilli are
resistant to immune attack as they are transformed into
a dormant stage with very low or nil metabolic and
replicative activity, however, a dormancy-related gene
set called DosR regulon continues to be expressed
during latent infection [99,101]. The exact physical and
metabolic nature and location of persistent tubercle
bacilli in the dormant state remains unknown. The
bacilli can remain dormant for the entire life of the host
without ever causing active disease or they may cause
disease several years or even decades later [109,110].
Impaired immunity due to exhaustion or suppression of
T cells results in resuscitation of M. tuberculosis from a
dormant to a metabolically active stage leading to active
TB disease (reactivation TB) [25,101]. However, the risk
of developing reactivation TB disease is highest during
the first two years after infection with M. tuberculosis
[109,113]. Similarly, reactivation TB in
immunocompetent individuals immigrating from TB endemic countries
to low TB incidence countries also occurs usually within
the first two years of their migration [6,9,113,114].
Based on these observations and some recent
experimental data, a dynamic model of latent infection has
been proposed recently in which endogenous
reactivation as well as damage response occurs constantly in
immunocompetent individuals .
The model suggests that during initial stages
(developing granuloma) of infection, M. tuberculosis grow well
inside phagosome and then escape from phagosome/
phagolysosome and are released in extracellular milieu
due to macrophage necrosis [69,70,116,117]. Some of
the extracellular bacilli stop replicating due to hypoxic
and acidic environment, nutrient limitation (conditions
that mimic stationary bacterial cultures) and presence of
bactericidal enzymes released from destruction of
immune cells, even before an effective immune response
is fully developed. With the development of an effective
immune response, the actively growing bacilli are easily
killed, however, the metabolically inactive,
non-replicating (dormant) bacilli resist killing and may survive .
The model also assigns an important role to foamy
macrophages that emerge during chronic inflammatory
processes (such as TB) due to phagocytosis of cellular
debris rich in fatty acids and cholesterol in the
dissemination and/or waning of infection. The model suggests
that as foamy macrophages phagocytose extracellular
non-replicating lipid-rich M. tuberculosis along with
other cellular debris, the bacilli are not killed due to
their non-replicating, metabolically inert (dormant)
state. At the same time, tubercle bacilli also do not
grow in the intracellular environment as the
macrophages are now activated [118-120]. As the foamy
macrophages containing non-replicating bacilli drain
from lung granuloma towards bronchial tree, they lodge
M. tuberculosis into a different region of lung
parenchyma due to aerosols generated by inspired air and the
bacilli get another chance to begin the infection process
at this new location [115,118,119,121]. In this
infectioncontrol of growth-reinfection process, bacilli getting
lodged in the upper lobe may have the chance to cause
cavitary lesion. This is due to higher oxygen pressure in
upper lobes that can support rapid extracellular bacillary
growth resulting in bacillary concentration that can not
be controlled by the optimum immune response
mounted by the host. The subsequent much stronger
inflammatory response leads to tissue destruction,
liquefaction and extracellular bacillary growth which
amplifies the response further and causes cavitation [115,116].
The dynamic infection model, involving drainage and
destruction of non-replicating bacilli in the stomach
over a period of time, proposes slow clearance (waning)
of latent infection in a sub-set of infected individuals
who are not at risk of reinfection. A recent study carried
out in Norway, a country with a low risk of active
transmission of infection or reinfection, has shown that rates
of reactivation TB, among patients previously exposed
to M. tuberculosis, have progressively declined over the
last several years . Furthermore, the prevention of
reinfection by bacilli resuscitated from dormancy by
isoniazid, during infection-control of growth-reinfection
cycles, also explains how therapy for only nine months
with a single drug, effective only against actively dividing
bacilli, is highly effective for a latent infection sustained
by non-replicating bacilli that can presumably survive
during the lifetime of the host .
Diagnosis of latent M. tuberculosis infection
Despite the fact that control and management of TB in
many low TB incidence countries is centered around
the identification and subsequent treatment of
individuals latently infected with M. tuberculosis (LTBI),
actual identification of LTBI in human subjects is
presently not feasible [123,124]. The current diagnostic
tests (such as the tuberculin skin test or more recently
developed T cell-based assays) are only designed to
measure the adaptive immune response of the host
exposed to M. tuberculosis, typically six to eight weeks
after exposure to the bacilli [123-126].
The tuberculin skin test (TST) measures cell-mediated
immunity in the form of a DTH response to a complex
cocktail of >200 M. tuberculosis antigens, known as
purified protein derivative (PPD) and the test result is
usually read as induration (in mm) recorded 48 to 72
hours after intradermal injection of PPD . The
criteria for a positive TST vary considerably and depend
on the inoculum and type of PPD preparation used in
the test. In the United States, 5 tuberculin units (TUs)
are generally used and the induration of ≥5 mm in
HIVseropositive or organ transplant recipient or in a person
in contact with a known case of active TB is considered
as positive . However, in foreign-born persons
originating from high TB incidence countries or persons at
higher risk of exposure to M. tuberculosis (such as
health care professionals), induration of ≥10 mm is
regarded as positive TST . In most European
countries, 2 TUs are used and the induration of ≥10 mm in
immunocompetent adults is considered as positive. In
the United Kingdom, 10 TUs are used and the
induration of 5-15 mm in BCG unvaccinated and ≥15 mm in
BCG vaccinated immunocompetent adults is considered
as positive [123-126]. Skin test reaction over 20 mm is
usually due to active disease; however, a negative skin
test in an active TB patient may also result from anergy
or incorrect administration of the test or improper
storage of the test reagents, thus compromising the
sensitivity of the test [9,104,127,128]. Skin testing is most
suitable for detecting M. tuberculosis infection in
developing countries where >80% of the global TB cases
occur, as it does not require extensive laboratory
facilities and health care workers are already familiar with
administering and reading skin tests. However, TST has
several inherent problems as the antigens present in
PPD are also present in the vaccine strain M. bovis BCG
and several environmental mycobacteria. Hence, TST
has lower specificity as the test can not differentiate
between infection with M. tuberculosis, prior vaccination
with M. bovis BCG or sensitization with environmental
mycobacteria [9,104,127,129,130]. Furthermore,
sensitivity of TST is limited in immunocompromised
individuals due to anergy. These factors have compromised
the sensitivity and specificity of tuberculin skin test for
the diagnosis of LTBI.
Highly sensitive and more specific tests for the
diagnosis of LTBI have been developed recently as a result
of advances in genomics and immunology. The
availability of complete genome sequences of M. tuberculosis
and other Mycobacterium spp. and subtractive
hybridization-based approaches identified RD1, a genomic
region that is present in all M. tuberculosis and
pathogenic M. bovis strains but is absent in all M. bovis BCG
vaccine strains and most of the environmental
mycobacteria of clinical relevance [13,64,65]. Two of the RD1
encoded proteins, ESAT-6 and CFP-10 are strong T cell
antigens [62,63]. Early studies in animals showed that
DTH skin responses to ESAT-6 and CFP-10
discriminated between animals infected with M. tuberculosis
from those sensitized to M. bovis BCG or environmental
mycobacteria . The rESAT-6 obtained from E. coli
is also biologically active and was successfully used as a
skin test reagent for the diagnosis of tuberculosis
infection in humans in phase I clinical trials [132,133]. The
sensitivity of rESAT-6 has been enhanced further by
combining it with CFP-10 and the ESAT-6/CFP-10
fusion protein was found to be as sensitive as PPD in
predicting disease in M. tuberculosis-infected guinea
pigs . It is expected that rESAT-6/CFP-10 fusion
protein could probably replace PPD as skin test reagent
for identifying individuals with LTBI.
Other cell mediated immunity-based assays have also
been developed. The in vitro T cell-based
interferongamma (IFN-g) release assays (IGRAs) were developed
based on the principle that T cells of individuals
sensitized with M. tuberculosis antigens produce high levels
of IFN-g in response to a reencounter with these
antigens . Initially IGRAs used PPD as the stimulating
antigen, however, it was subsequently replaced by two
M. tuberculosis-specific T cell antigens; ESAT-6 and
CFP-10 and the assays were found to be sensitive and
specific for detection of active
pulmonary/extrapulmonary TB as well as latent infection [136-140].
Two commercial IGRAs, whole blood, ELISA-based
QuantiFERON-TB Gold (Cellestis Ltd., Carnegie,
Australia) and peripheral blood mononuclear cell (PBMC) and
enzyme-linked immunospot (ELISPOT)
technologybased T-SPOT.TB (Oxford Immunotec, Oxford, UK)
tests were subsequently developed and approved by Food
and Drug Administration (FDA) for detecting latent
infection. The first-generation QuantiFERON-TB Gold
test was based on stimulation of T lymphocytes with
PPD and measurement of IFN-g production . The
enhanced QuantiFERON-TB Gold assay subsequently
used ESAT-6 and CFP-10 proteins as stimulating
antigens. The first-generation T-SPOT.TB used ESAT-6 and
CFP-10 proteins as stimulating antigens and detected
Tcells themselves . These commercial tests have
undergone further improvement since their inception.
The newer version of the QuantiFERON-TB Gold assay
is called QuantiFERON-TB-Gold-In-Tube (QFT-G-IT)
(Cellestis Ltd., Carnegie, Australia) that uses ESAT-6 and
CFP-10 and TB7.7 (corresponding to Rv2654 )
peptides as antigens. The newer version of T-SPOT.TB also
uses peptides of ESAT-6 and CFP-10 instead of whole
ESAT-6 and CFP-10 proteins as antigens (Oxford
Immunotec, Oxford, UK).
The performance of both QFT-G-IT and T-SPOT.TB
tests have been evaluated extensively with/without
headto-head comparison with TST and several systematic
reviews are available for their performance in different
settings [123-126,142-144]. Similar to TST, a major
limitation of both IGRAs is their inability to distinguish
LTBI from active TB disease. This may be particularly
important in high TB incidence countries in which
latent infection is widespread and reinfection happens
frequently and in immunocompromised individuals
(such HIV-seropositive subjects) and children due to
subclinical disease presentation [123,124,126]. However,
IGRAs have better specificity (higher that TST) as they
are not affected by prior BCG vaccination since the
antigens used in these assays are not present in M. bovis
BCG and cross reactivity with environmental
mycobacteria is less likely [123-125]. Furthermore, based on
limited data in immunocompromised individuals, the
sensitivity of IGRAs, particularly for T-SPOT.TB, is also
higher than TST . However, the clinical
performance of these tests has been variable in different
settings around the globe due to differences in spectrum
and severity of TB cases and proportion of
HIV-coinfected individuals included in various studies [123,126].
In low TB incidence countries, screening for LTBI
aims to identify individuals at higher risk of progression
from latent infection to active TB disease. These include
all recently infected individuals (close contacts of active
pulmonary TB index case), recent immigrants from high
TB incidence countries and persons with suppressed
(such as HIV coinfected) or immature (such as very
young children) cellular immune systems [123,126,142].
Previous data on natural history of TB suggest that after
exposure to M. tuberculosis, 5-10% of infected
individuals develop active TB disease within the first 2 years
of initial infection [109,113]. In people with a robust
immune system, another 5-10% individuals develop
active disease during the remainder of their lives while
in immunocompromised individuals, the risk is much
higher [123,124]. Thus, diagnosis and treatment of LTBI
will be most effective if it is specifically directed to
those individuals with the highest risk of progression
from LTBI to active disease such as recently exposed
individuals, young children and HIV-infected and other
The current cumulative evidence (summarized in
several reviews and meta-analyses) [123-126,142-144]
suggest that the performance of the two (ELISA-based and
ELISPOT-based) formats of IGRAs are nearly
comparable in predicting development of active disease in
immunocompetent individuals. However, the agreement
between IGRAs and TST is generally poor due to
falsepositive TST results in BCG vaccinated subjects. The
clinical relevance of a positive TST result is usually poor
(i.e. unable to predict which patients will develop active
TB disease in the near future) and sensitivity as well as
specificity are influenced by the different cut-off values
used in different settings. However, the value of negative
TST result in predicting no further development of
active disease in human subjects presumably exposed to
M. tuberculosis is fairly high (negative predictive value).
On the other hand, the predictive value of positive
IGRA results for the development of active TB is usually
better than that of TST while the predictive value of a
negative result is very high in immunocompetent
individuals, particularly if the TST is also negative [123-126].
The TST is often negative in immunocompromised
individuals and its performance is also influenced by the
immunosuppressing conditions while the sensitivity of
IGRAs is generally better than TST and the
experimental conditions (particularly in T-SPOT.TB assay) can be
easily adjusted for testing immunocompromised
A major problem associated with IGRAs is the
occurrence of indeterminate results that seem to arise mostly
due to cellular immune suppression and occur more
frequently with the ELISA-based method than with
ELISPOT test or discordant results if both, TST and a
blood test are performed [123,124]. This is further
compounded by the differences that exist in the manner in
which these tests are applied for the detection of latently
infected individuals in different settings. In the United
States and few other countries, national guidelines
advocate up-front use of a blood test (IGRA) as a direct
replacement for TST in all groups of subjects .
Due to higher sensitivity of IGRAs, it is likely that some
individuals who are positive for a blood test but who
may have been TST negative (if the test was performed)
are unnecessarily treated. On the contrary, in the United
Kingdom and other European countries, initial screening
is performed with TST except in individuals in whom
TST is unreliable (young children, HIV-seropositive and
other immunosuppressed individuals) [124,146]. For the
latter grouping and for TST-positive individuals at
higher risk of developing active disease, a blood test is
recommended for confirmation of a presumed infection.
Thus, it is also probable that a TST-negative subject
who may have been IGRA positive will not be identified
as having LTBI and will, therefore, not receive
treatment. Consequently this apoproach, though supposedly
more economical, may result in undertreatment of some
individuals with LTBI [123,124]. A discordant result
(TST negative but IGRA positive) in an
immunocompetent individual should be repeated after 3 months and
should be treated for LTBI if IGRA still remains positive
(a negative IGRA on repeat testing may signify a transient
M. tuberculosis infection that was quickly cleared) .
However, a similar result in an immunocompromised
individual should be carefully evaluated as in this setting,
any positive result may be significant.
Although both, TST and IGRAs cannot distinguish
between LTBI and active TB disease in
immunocompetent adults [123,126], however, in high-risk individuals
with immunosuppressive conditions and children,
IGRAs may help in the investigation of active disease as
adjunctive diagnostic tests, particularly if specimens
(such as bronchoalveolar lavage, cerebrospinal fluid)
from the suspected site of infection rather than blood is
used for the diagnostic assay [147-149]. While the
results of IGRAs exhibit better correlation with
surrogate measures of exposure to M. tuberculosis in low TB
incidence countries, however, their performance is
generally sub-optimal in countries with a high TB incidence
[123-126,143,144,150]. Application of targeted
tuberculin skin testing and IGRAs to identify latently infected
individuals and their treatment for LTBI has greatly
helped in lowering the incidence of TB in rich, advanced
countries [128,138,140,144,151]. Previous studies have
shown that majority of active disease cases in low or
low-intermediate incidence countries in immigrants/
expatriates originating from TB endemic countries
occur as a result of reactivation of previously acquired
infection mostly within two years of their migration
[6,9,113,114,140]. Some other low-intermediate TB
incidence countries which contain large expatriate
populations originating from TB endemic countries are also
evolving similar strategies for controlling TB [152-157].
Another variation of conventional cell mediated
immunity-based assays (IGRAs) has also been developed
by using flow cytometry . Although flow cytometric
approach uses smaller blood volume (<1 ml), the assay
will have limited utility in much of the developing world
due to the high cost of flow cytometers and the need
for technically experienced personnel. The detection of
significant levels of antibodies to some M.
tuberculosisspecific proteins has also been noted in contacts of TB
patients (latently infected individuals) as well as in
patients with active TB disease but not in healthy
subjects [159-162]. However, antibody-based methods are
only experimental and are not used in clinical practice
for the detection of LTBI.
Treatment of latent M. tuberculosis infection
Tracing contacts of infectious pulmonary TB cases
(sputum smear-positive) for exposure to tubercle bacilli
leading to latent M. tuberculosis infection (LTBI) and
treatment of latently-infected individuals at high risk of
progressing from latent infection to active disease has
proven extremely effective in the control of TB in the
United States and other low TB-burden countries
[128,151,163]. Treatment of LTBI in infected persons
substantially reduces the likelihood of activation of
dormant infection and subsequent development of active
TB disease (Figure 1). The American Thoracic Society
(ATS) and Centers for Disease Control and Prevention
(CDC) issued guidelines in 2000 for the treatment of
LTBI which were also endorsed by the Infectious
Diseases Society of America and American Academy of
Pediatrics . An update to these guidelines was
published in 2005 that also included recommendations for
pediatric subjects . The treatment options currently
available for LTBI are summarized in Table 2.
The standard regimen for the treatment of LTBI in
United States and Canada is daily self-administered
therapy with isoniazid (INH) for nine months based on
clinical trial data but the duration of treatment can be
reduced to 6 months for adults seronegative for
HIVinfection [128,164]. The International Union Against
Tuberculosis (IUAT) recommends daily therapy with
INH for 12 months as it is more effective than the
6month course (75% vs. 65%) . The preferred
duration of treatment for most patients with LTBI in the
United States and European countries is 9 months since
clinical trial data showed that the efficacy of 6-month
regimen is reduced to 60% while 12-month regimen is
advocated for individuals at higher risk of developing
active disease [123,166]. According to the CDC
guidelines, the frequency can also be reduced from daily
therapy to twice weekly therapy with increased dosage of
INH, however, the twice weekly regimen must be given
as directly observed treatment (DOT) . Inclusion of
DOT adds a substantial additional expense to the
treatment strategies. The efficacy of INH treatment in
preventing active TB exceeds 90% among persons who
complete treatment . However, the overall
effectiveness of these regimens is severely limited as the
completion rates in clinical settings have been rather
low, ranging from 30% to 64% only [167-169].
Completion rates in other settings have been even lower .
Although INH is tolerated fairly well by most of the
individuals, there is a risk of hepatic toxicity in selected
populations. Studies have shown that 10% to 22% of
participants taking INH for LTBI have at least one
episode of elevated serum transaminase levels. Although
the rates of clinically significant hepatitis were much
lower (< 2%), the risk and severity increased with age
and concomitant alcohol consumption [171-173]. INH
can also cause peripheral neuropathy but the risk can be
lowered by concomitant use of pyridoxine (vitamin B6)
. Poor adherence due to the long duration of
treatment and concerns for hepatotoxicity in selected patient
populations resulted in development of shorter and
more effective treatment options for LTBI [128,164].
The ATS and CDC guidelines also included 4 months
of rifampicin (RMP) alone or 2 months of RMP and
pyrazinamide (PZA) as acceptable alternatives for the
treatment of LTBI . The RMP alone is
recommended for persons intolerant to INH, close contacts of
TB cases in which the isolate of M. tuberculosis is
resistant to INH or INH resistance is suspected due to the
origin of foreign-born persons from countries where
INH resistance rates are high [128,175,176]. There are
several advantages with 4 month daily therapy with
RMP such as lower cost, higher adherence to treatment
and fewer adverse reactions including hepatotoxicity
[151,169,177-180]. However, treatment with RMP alone
is not recommended for HIV-seropositive persons on
concomitant anti-retroviral therapy as this may lead to
the development of acquired rifamycin resistance
[164,181,182]. Furthermore, active disease in an
HIVinfected individual should be ruled out first since
monodrug therapy in an undiagnosed active TB disease case
may also lead to RMP resistance. However, active TB
disease is more difficult to exclude in HIV-infected
individuals as they are less likely to have typical features of
pulmonary TB and extrapulmonary TB occurs more
Table 2 Currently available drug regimens for the treatment of latent tuberculosis infection
Duration of treatment
Preferred regimen by CDC
For HIV seronegative only
For HIV seronegative only
Preferred regimen by IUAT
Good alternative option
Higher risk of hepatotoxicity
Higerh risk of hepatotoxicity
For LTBI with INHr strain in HIV seronegative subjects
INH, isoniazid; RMP, rifampicin; PZA, pyrazinamide; RPE, rifapentine; DOT, directly observed treatment; 2/Wk, twice weekly; 1/Wk, once weekly; CDC, Center for
Disease Control and Prevention; HIV, human immunodeficiency virus; IUAT, international Union Against Tuberculosis; LTBI, latent tuberculosis infection
frequently [6,183,184]. The regimen of RMP alone is
also not suitable for patients with other underlying
conditions such as diabetes [185,186].
Treatment of LTBI with RMP + PZA for 4 months is
another alternative choice that was advocated by ATS
and CDC guidelines in 2000 . Although initial
studies with 2 months of RMP + PZA in HIV-infected
persons were reported to be as effective and safe as INH
treatment [187,188], several cases of severe liver injury
and/or death were reported subsequently with the
RMP + PZA regimen resulting in revision of ATS/CDC
recommendations in 2003 . The revised guidelines
advocated that 2 months of RMP + PZA regimen should
not generally be offered to HIV-seronegative or
HIVseropositive individuals [163,164]. A meta-analysis
involving six clinical trials comparing the effectiveness of 2
months of RMP + PZA with 6 or 12 months of INH
treatment showed that RMP + PZA regimen was
associated with increased risk of hepatotoxicity in HIV
seronegative persons while the results for HIV-infected
persons were inconclusive . However, when the
results of 2 months of RMP + PZA were compared with
6 months of INH treatment without supplementation
with pyridoxine in HIV-infected persons, the data
showed no significant differences in hepatotoxicity in
the two sub-groups. The results of some studies suggest
that 2 months of RMP + PZA regimen may also be
considered when other regimens are unsuitable and
monitoring of liver function tests is feasible [191,192].
Other options that have been tested or are under
evaluation for the treatment of LTBI include 3 months of
INH + RMP given daily or twice weekly under DOT
and 3 months of INH + rifapentin (RPE) given once
weekly. The 3 months of INH + RMP regimen has been
tested mostly in the United Kingdom. A meta-analysis
of five studies carried out in both HIV-infected and
HIV-seronegative individuals as well as two subsequent
studies have shown that the 3 month of INH + RMP
treatment is well tolerated and is as effective and safe as
6 to 12 months of INH treatment alone [193-195]. The
longer half life of RPE, approved by U. S. Food and
Drug Administration (FDA) in 1998 for the treatment of
TB, has allowed once weekly dosing of INH + RPE for
the treatment of LTBI . One small study comparing
once-weekly INH + RPE for 3 months with daily RMP +
PZA for 2 months reported fewer discontinuation of
treatment due to hepatotoxicity in the INH + RPE arm
compared to the RMP + PZA arm even though the risk
of developing active TB was nearly same in both the
groups . A large multi-center study is currently
being conducted by the Tuberculosis Trials Consortium
of the CDC to determine the efficacy of once weekly
dosing of INH + RPE in preventing active disease
among high-risk individuals with LTBI. However, the
A major concern that has arisen recently is the threat of
latent infection in a person exposed to a source case
infected with multidrug-resistant strain of M.
tuberculosis (MDR-TB). As nearly 440 000 cases of MDR-TB
corresponding to nearly 5% of all incident TB cases
occurred in 2008 , this concern is likely to attract
greater attention in the near future. Only scant
information is available in this setting as there have been no
randomized controlled trials to assess the effectiveness
of specific regimens . A 6 to 12 month regimen of
a fluoroquinolone + pyrazinamide or ethambutol +
pyrazinamide is recommended by CDC. However, the
effectiveness and optimal duration of these regimens is
largely unknown as they are very poorly tolerated .
The newer drugs that are in different stages of
development may offer better alternatives for the treatment of
both, active TB disease as well as LTBI.
The new generation fluoroquinolones such as
moxifloxacin have excellent (bactericidal) activity against M.
tuberculosis and may be more effective in the treatment
of LTBI than older drugs of the same class [200,201]. In
experimental animal model of latent infection, the once
weekly regimen of rifapentine + moxifloxacin for 3
months was found to be as effective as daily therapy
with isoniazid for 9 months . The PA-824, a
nitroimidazo-oxazine, is another promising compound that is
active against MDR-TB strains and is also active against
non-replicating persistent bacteria, making it an ideal
drug candidate for the treatment of LTBI. The
treatment regimen containing PA-824, moxifloxacin, and
pyrazinamide was highly effective in murine model of
tuberculosis . The OPC-67683, a
nitroimidazo-oxazone, is another promising new compound that shows
promising results against tuberculosis in mice . A
diarylquinoline (R207910 also known as TMC207) has
shown more potent early bactericidal activity than INH
during early phase of infection and higher bactericidal
activity late in infection than RMP alone and thus may
provide another option for the treatment of LTBI
[205,206]. Another promising drug is SQ109
(1,2-ethylenediamine) that is structurally related to ethambutol but
is more potent [207,208]. It is expected that some of
these new drugs will provide additional options for the
treatment of LTBI in the near future.
Another approach that is actively being pursued for
controlling development of active disease in persons
with LTBI is development of novel vaccines that may
prevent TB disease reactivation by efficiently containing
the pathogen in a latent state in infected individuals
[209-211]. More than 10 vaccine candidates have
entered clinical trials in the past few years . Two of
these vaccine candidates are recombinant M. bovis BCG
constructs designed to improve the antigenicity and/or
immunogenicity of the current BCG vaccine [212,213].
Another seven subunit vaccines are being tested in
clinical trials and are being used as booster vaccines
designed to reorient the immune response after priming
with recombinant BCG vaccines. Three of the subunit
vaccines are incorporated in viral carriers while the
other four subunit vaccines are being delivered through
adjuvant formulations [209,214-216]. The recombinant
BCG and booster subunit vaccines are designed to be
given prior to M. tuberculosis infection to sustain latent
infection and either prevent or delay the reactivation of
latent infection by inducing a memory T cell response
that resists exhaustion and suppression . Other
vaccine candidates under development include further
modifications such as inclusion of dormancy-regulated
genes to improve the efficacy of BCG replacement
vaccine candidates for post-exposure vaccination of latently
infected individuals (Figure 1) [101,209]. A drawback of
the above vaccines is that they prevent or delay the
reactivation of dormant infection but do not eradicate
the pathogen. However, attempts are now underway to
combine the antigens of metabolically active (such as
secreted proteins) and dormant (such as
dormancyregulated genes) state of M. tuberculosis in both, the
recombinant BCG and subunit booster vaccines to
achieve sterile eradication of the pathogen .
Infection with M. tuberculosis begins with the
phagocytosis of tubercle bacilli by antigen-presenting cells in
human lung alveoli. This sets in motion a complex
infection process by the pathogen and a potentially
protective immune response by the host. M. tuberculosis
has devoted a large part of its genome towards functions
that allow it to successfully establish progressive or
latent infection in majority of infected individuals. The
failure of immune-mediated clearance is due to multiple
strategies adopted by M. tuberculosis that blunt the
microbicidal mechanisms of infected immune cells and
formation of distinct granulomatous lesions that differ
in their ability to suppress or support the persistence of
viable M. tuberculosis (LTBI). A positive tuberculin skin
test or T cell-based interferon-g release assay in a
person with no overt signs of active disease indicates LTBI
and requires treatment of individuals particularly those
at the highest risk of progression from LTBI to active
disease such as recently exposed individuals, young
children and HIV-infected and other immunocompromised
subjects. Standard treatment regimen for LTBI is daily
therapy with isoniazid for nine months. New drugs/drug
combinations as well as novel vaccine approaches are
being developed for eradication of latent infection in
exposed individuals. Identification and treatment of
latently infected individuals has greatly helped in control
of TB in rich, advanced countries and similar
approaches hold great promise for other countries with
low-intermediate rates of TB incidence.
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