Review Articles

The Emergence of Extensively Drug-Resistant Tuberculosis: A Global

Health Crisis Requiring New Interventions: Part II: Scientific Advances
that May Provide Solutions
Jerrold J. Ellner, M.D.

Abstract
The need for new tuberculosis (TB) diagnostics has never been greater as TB in the HIV-infected is often sputum smear negative or
extrapulmonary and may progress rapidly unless diagnosed and treated appropriately. In addition, the empirical treatment of patients with
drug-resistant TB leads to the acquisition of additional resistance. Fortunately there is a robust and adequately funded developmental
pipeline including investigational rapid diagnostics that may replace smear, culture, and drug susceptibility testing. The dogma that drug
resistance usually develops as a consequence of patient nonadherence has never been entirely plausible. Recent observations indicate
that certain mutations in drug resistance genes promote the acquisition of additional resistance. Further, Mycobacterium tuberculosis
(MTB) may demonstrate tolerant phenotypes due to induction of a multidrug-resistant like pump. It will be difficult to “treat our way”
out of the problem of extensively drug-resistant (XDR)-TB without access to new interventions. Vaccines in development offer a distant
hope. Promising new therapeutics in clinical trials may shorten the duration of treatment of TB, which will lessen the development of
drug resistance or provide potent new and MTB specific agents that should be effective in treatment of XDR-TB.
Keywords: tuberculosis, drug resistance, multidrug-resistant TB, extensively drug-resistant TB, anti-tuberculous drugs, anti-
tuberculous vaccines, TB diagnostics

Introduction
This is the second of a two-part review of extensively drug- resistant
(XDR) tuberculosis (TB). The first part, published in Volume 1
Issue 3 of Clinical and Translational Sciences, considered the
clinical and public health issues posed by multidrug-resistant
(MDR)-TB and XDR-TB. The second section will examine the
need for new diagnostics, developments in diagnostics, advances
in understanding mechanisms of resistance, and new vaccines
and therapies.
The Need for New Diagnostics
Approximately 95% of TB incidence and 99% of TB mortality
occur in resource-constrained environments, thereby adding a
number of economically related obstacles to assuring access of TB
suspects to appropriate TB diagnostics. In most resource-limited
settings, the diagnosis of TB is based on clinical algorithms often,
but not invariably, supported by sputum microscopy. Reference
laboratories with the capacity to perform culture and drug
susceptibility testing (DST) exist in many TB endemic countries,
but access to these services usually does not extend beyond central
reference laboratories and research settings.
The problem of inadequate laboratory infrastructure
has become more of a public health concern because of the
comorbidity of HIV infection and the spread of drug-resistant
Mycobacterium tuberculosis (MTB). HIV-TB coinfection is
associated with increasing prevalence of TB cases and an
increasing proportion that are sputum-smear negative and/or
extrapulmonary in nature. Precise diagnosis is more difficult in
this setting, also known as “paucibacillary” TB. Diagnosis of TB
is, however, also more critical to patient outcome because HIV-
associated TB is associated with rapid progression of TB disease
and a high case-fatality rate. As discussed in the first part of this
review, drug-resistant TB including MDR and XDR-TB now has
been documented worldwide. Even in countries with a relatively
low prevalence of drug resistance, such as Uganda, a significant
proportion (over 12%) of retreatment cases show acquired drug
resistance.1 The current approach to management of retreatment
cases in most TB endemic countries is to administer an enhanced
treatment regimen that includes an injectable antibiotic in the
intensive phase. The general lack of access to DST is problematic
from two standpoints. First, those TB patients with fully drug-
susceptible isolates are overtreated and exposed needlessly to
more expensive and toxic regimens. Second, patients with drug-
resistant TB, particularly MDR-TB, receive an ineffective regimen
likely to lead to acquisition of additional drug resistance, even
to XDR-TB. Quite clearly, empirical management of TB patients
at risk for MDR-TB with DST for first line drugs promotes
emergence of resistance to first line drugs. Isolates that are MDR
at the start of treatment show amplification of drug resistance
with loss of susceptibility to ethambutol and pyrazinamide.1 There
also is evidence that management of MDR-TB with an empirical
“DOTS-plus” regimen, which includes second-line drugs without
relevant DST, contributes to the emergence of additional drug
resistance and ultimately XDR-TB.2 The delay in diagnosis and
initiation of appropriate therapy in drug-resistant TB also leads
to prolonged nosocomial transmission as exemplified by the
XDR-TB outbreak in Tugela Ferry.3
Development of Improved Diagnostics
Culture currently is the precursor to DST. As recently reviewed,
access to culture is rare in TB endemic countries, except for the
Russian Federation, Brazil, and South Africa.4 Access to quality-
controlled DST is even more limited. Of the 22 highest TB burden
countries, fewer than 50% have at least three laboratories capable
of DST. Technologies based on automated culture in liquid
media using light emission reflective of bacterial metabolism
as an endpoint (MGIT system) can more than halve the time
for the diagnosis of TB. DST performed in liquid medium is
available in 2–4 weeks rather than the standard 8–18 weeks when

New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA.
Correspondence: JJ Ellner (jerroldellner@yahoo.com)
DOI: 10.1111/j.1752-8062.2008.00086.x

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solid medium is used. If DST is performed with MGIT directly
from sputum of smear-positive cases, results are available in
1–3 weeks. In order to increase availability of DST, major efforts
must be placed on developing the laboratory infrastructure.
Demonstration projects planned for Zambia, Brazil, and South
Africa will expand access to liquid culture in these countries.
Another approach is to attempt a technologic “fix” in which the
need for culture ultimately will be bypassed. The Foundation for
Innovative Diagnostics (FIND) has assumed a pivotal coordinating
role in the development, evaluation, and implementation of
new TB diagnostics and at present has 35 diagnostics in the
“pipeline”. These fall within the general categories of growth-
based detection, direct visualization, volatiles detection, antigen
detection, antibody detection, molecular detection, speciation,
and diagnosis of latent TB infection.
The World Health Organization recently approved an
investigational diagnostic, the Hain genotype MTBDR plus assay
(a line probe assay), for use in the diagnosis of MDR-TB. With a
turn-around time of 1 week, it allows determination that sputum
contains MTB-complex organisms and provides DST for isoniazid
and rifampin. The Hain test as it is called (developed by Hain
Lifesciences, Hardwiesenstr, Nehren) requires molecular training
of technicians and must be performed in a reference laboratory.
The test is an open-tube method so that there is a risk of amplicon
contamination and false positive tests. In a study from South Africa,
the Hain assay was performed on sputum from 536 TB patients
(28% HIV-TB coinfection rate) at increased risk of drug resistance.5
Overall, 97% of smear-positive specimens gave interpretable results
within 1–2 days, using the Hain molecular assay. Sensitivity,
specificity, and positive and negative predictive values were 98.9%,
99.4%, and 97.9% and 99.7%, respectively, for detection of rifampin
resistance; 94.2%, 99.7%, and 99.1% and 97.9%, respectively, for
detection of isoniazid resistance; and 98.8%, 100%, and 100%
and 99.7%, respectively, for detection of multidrug resistance
compared with conventional results. The assay also performed well
on specimens that were contaminated on conventional culture and
on smear negative, culture positive specimens. Large demonstration
projects using the Hain assay are planned in South Africa, Uganda,
and Russia, Uzbekistan, and Nepal.
Two of the most promising molecular detections assays
in development will be discussed in detail: the Eiken TB
Loop Mediated Isothermal Amplification (LAMP) Test and
the Cepheid Xpert MTB test. The LAMP test for TB could
well replace microscopy. The technology uses isothermal
amplification, which removes the need for a thermocycler, and
functions in a closed tube, which reduces the chance of false
positive results from work-space contamination with amplified
DNA. It is a very rapid assay, with results available in less than
45 minutes. Importantly, it generates results based on turbidity
and fluorescence that can be seen without a microscope and
does not require molecular training of technicians (Figure 1).
In studies of 725 sputum specimens from 380 TB suspects, the
sensitivity of the LAMP assay was 98% in acid fast bacilli (AFB)
smear positive cases and 49% in smear negative but culture
positive cases.6 Specificity in culture positive cases was 99%.
These data are very promising and could allow real-time point-
of-care (POC) diagnosis while the patient is waiting in clinic.
LAMP may have particular value in populations with a high
prevalence of HIV infection because of the greater frequency
of smear negative cases.
The GeneXpert (Cepheid, Sunnyvale, California, USA) is
a technology platform for automated specimen processing and
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nucleic acid amplification-based detection of pathogens and
their genotypic characteristics. Closed-cartridge amplification
minimizes the risk of amplicon contamination, and different
cartridges can be used to detect different pathogens.
Xpert MTB is a recently developed TB-specific application,
designed for the GeneXpert platform to detect MTB as well as
rifampin resistance-conferring mutations directly from sputum,
in an assay having a start-to-finish run time of 100 minutes. The
ease of use and target performance specifications for Xpert MTB
would allow it to be used in POC or district level laboratories, as a
replacement for culture, detecting both smear-positive and smear
negative TB and screening for rifampin resistance as a marker of
MDR-TB. In preliminary studies, the sensitivity in AFB smear
positive cases has been 98–100% and the sensitivity in smear
negative culture positive cases 72% (Alland D, unpublished data).
The specificity has been 100%. The sensitivity and specificity for
detection of rifampin resistance has been 100%. The Xpert MTB
test should be extremely useful when applied to populations with
a high level of TB drug resistance. Because cartridges are being
developed for HIV viral load testing, the GenExpert device may be
particularly applicable in areas in which coinfection is frequent.
The history of the development of TB diagnostics provides
useful insights into the implementation obstacles that remain for
these and other investigational diagnostics. PubMed indicates
that over 1,900 articles on serodiagnostics for TB have appeared
in the last four decades. Confidence in this approach and the
field of TB diagnostics, in general, has been undermined,
however, by inordinate variability in test performance due to
inadequate study designs, small sample sizes, poorly characterized
clinical populations, inadequate quality-control of laboratory
endpoints, lack of a standard prototype, and researcher bias.7,8
A standardized approach to the reporting of trials of diagnostics
(STARD) has been proposed as a means of improving the accuracy
and completeness of reporting so that the reader can evaluate
potential reproducibility as well as bias.9 Another issue related
to the implementation of new TB diagnostics is illustrated by the
assays based on DNA amplification (e.g., Gen-Probe Amplified
MTB Direct Test), which currently are approved in the US. These
have had little clinical use because of their expense and limited
impact on TB management. Future studies of TB diagnostics
will need to avoid pitfalls in design and execution and should be
accompanied by cost-effectiveness analysis.
Recent Advances in Understanding Drug Resistance in TB
Most retreatment cases of TB remain fully drug susceptible. Further,
drug resistance may develop while patients are being treated with
combination regimens that are active against the isolate. It is
not entirely clear, therefore, why certain patients develop drug
resistance whereas others do not. In the past, the patient has often
been blamed for “poor adherence” with therapy. This premise
needs to be examined more carefully. Missing doses of a multidrug
anti-TB regimen, the most common form of nonadherence, is not
likely, in itself, to promote the development of drug-resistant TB;
rather, periods of “monotherapy” early in the course of treatment,
when the bacterial burden is high, is more likely to select resistant
isolates. This occurs, in effect, when a patient with drug-resistant
MTB is treated with regimens that would be effective against drug
susceptible organisms. It is not clear how often or why a patient
by virtue of their own nonadherence would selectively ingest
some but not all prescribed drugs. Cavitary disease is known to
be a risk factor for the development of resistance. Certainly, the
high bacterial burden and poor drug penetration of cavities as
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Figure 1. Visual detection of LAMP product under UV light. From left to right, tubes 1, 2, 7, and 8 are positive;
3, 4, 5, and 6 are negative. Reprinted from Ref. 6 with permission.
well as variable absorption of drugs may be important variables
in distinguishing patients who develop resistance from those
who do not.
Four recent observations require rethinking of the mechanism
of development of MDR and XDR-TB. They suggest, in toto,
that the strain of MTB may influence the development of drug
resistance.
The first is based on study of the population genetics of
isoniazid-resistant mutations.10 Isoniazid-resistant mutations
differ in isoniazid-monoresistant MTB compared with those
with concurrent rifampin resistance. Mutations in the inhA
promoter were more common in monoresistant strains and
katG315 mutations in the MDR isolates. These findings support
the hypothesis that katG315 mutations maintain or increase the
fitness of monoresistant isolates and allows them to evolve to
MDR; likewise, inhA promoter mutations may attenuate the
strains so that they are less likely to acquire additional resistance.
Mutations in ahpC, inhA, and other sites in katG are associated
with rifampin resistance, whereas only katG315 mutations are
associated with ethambutol resistance. These findings indicate
that the evolution of MDR and XDR-TB strains is a complex and
dynamic process. Although spontaneous drug mutations may
be unlinked, this is not the case for the modifying interactions
between genes and drug-resistant phenotypes.
The second observation relates to the finding that a mutation
in the embB306 codon does not lead uniformly to ethambutol
resistance (45% of isolates with this mutation remain ethambutol
sensitive) but is associated with broad drug resistance and
increased propensity for transmission. Further, the transfer of
11
the embB306 mutation into MTB by allelic exchange increases
resistance to isoniazid and rifampin as well as ethambutol.12 The
basis for this is unknown; however, subinhibitory concentrations
of ethambutol increase cell wall permeability and thereby
susceptibility to hydrophobic antibiotics.13 The embB306 mutation
may similarly modulate cell wall permeability even in the absence
of ethambutol resistance. The result would be a “loss-of-synergy”
phenotype, possibly prone to become MDR and XDR.
Third, drug-resistant mutations may affect bacillary fitness.
The early finding of Mitchison14 was that the loss of the catalase
activity in isoniazid-resistant strains of MTB was associated with
loss of virulence. On the other hand, the katG315 mutation is
associated with epidemiologic clustering of isolates, a surrogate
for increased transmission.15 It is postulated that this mutation is
less likely than others to attenuate virulence.16 The preponderance
of Beijing clade isolates with katG315 mutations among MDR-TB
strains may relate to increased transmission due to the isonazid
17,18
resistance mutation, rather than other properties of the Beijing
82 Volume 2 • Issue 1
family, long thought to be hypervirulent.
The basis for the increased clustering, also
seen in embB306 mutations, is uncertain.
Fourth, drug tolerance also may play
a role in development of drug resistance
in MTB.19 Bacterial tolerance is defined as
delayed killing in vitro. There are inherent
differences in bactericidal activity of drugs
with rifampin > isoniazid-ethambutol >
ethambutol. Bacterial factors also affect
the bactericidal activity of drugs. In a
small study, MTB isolates with tolerance
to isoniazid or rifampin were more likely
to show persistence during therapy.18 The
molecular basis of tolerance to isoniazid
and ethambutol has been elucidated.20 Isoniazid and ethambutol
induce expression of genes iniA, iniB, and iniC. The iniA gene
confers tolerance for isoniazid and ethambutol, apparently
through its function as an efflux MDR-pump (Figure 2). Recent
studies indicate that the histone-like protein Lsr2 is involved in
transcriptional regulation of antibiotic-induced responses and
in MTB multidrug tolerance.21 This identifies a potential target
for intervention.
Approaches to combat XDR-TB
Certain aspects of the XDR-TB problem can be dealt with using
existing tools and approaches. Infection control measures are
effective in controlling nosocomial outbreaks of drug-resistant
MTB. Comprehensive infection control measures, however, are
costly and may not be feasible in resource-constrained settings;
however, even in these settings, use of personal respirators and
separation of susceptible hosts (HIV infection) from patients
with drug-resistant disease may be effective. Improved laboratory
services with access to DST for second-line drugs are an essential
component of effective management of patients with MDR-TB.
The administration of appropriate second-line drugs must be
monitored closely to assure for adverse events and to assure
compliance, which is critical to avoid acquisition of additional
drug resistance.
An improved TB vaccine would be the most effective means
of preventing the spread of XDR-TB. The STOP TB Working
Group on TB Vaccines estimates, however, that an expenditure of
$3 billion will be necessary to assure that a vaccine will be ready
for use in 2015. There are a number of issues besides the requisite
time and resources that must be considered in relationship to
TB vaccines. Most importantly, neither appropriate animals
Figure 2. Three-dimensional (3D) reconstruction of M. tuberculosis iniA
oligomers. (A) 2D crystals of hexameric MTB iniA. Inset: Projection of hexameric
iniA. (B) Surface representation of 3D reconstruction of the predominant iniA
oligomer obtained from single particle analysis shows C6 symmetry with threshold
at 438 kDa. Reprinted from Ref. 20 with permission.
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nor human correlates for protective immunity exist to increase
or predict the likelihood that a TB vaccine candidate will be
effective. The absence of immunologic correlates of protection
further complicates decisions on the dose and schedule for a
vaccine, as well as selection of adjuvants and formulations. In
areas with substantial prevalence of HIV infection, safety may be
a concern for live vaccines; further, vaccines may, in general, be
less efficacious in the presence of immunodeficiency. Lastly, the
efficacy of the current vaccine BCG in preventing TB meningitis
and disseminated TB in children means that a new vaccine
cannot be compared directly to placebo, at least for childhood
immunization. This will complicate trial design.
Several new vaccines have shown promising evidence for
protective efficacy in animal models and immunogenicity and
safety in humans.22 Four candidates currently are in human trials:
MVA85A; Mtb72f; AERAS 402; and Mycobacterium vaccae.
MVA85A is a recombinant modified vaccinia virus, expressing
a major secretory product of MTB—the 85A antigen. Mtb72f is
a combination of two immunogenic proteins Mtb 32a and Mtb
39a in two adjuvants ASO2A and ASO1B. It has recently been
reformulated and designated M72. AERAS 402 is a serotype 32
adenovirus, which is replication deficient and expresses three
MTB secretory proteins, 85A, 85B, and TB10.4. Mycobacterium
vaccae is a heat-killed soil organism. These candidates are in
various stages of Phase 1, 2, and 3 trials. They are intended as
protective vaccines or for use in a prime-boost strategy that will
include MTB-infected persons.
Development of New Drugs
New drugs will be necessary to treat the current and projected
future cases of XDR-TB, while awaiting routine administration
of a more effective TB vaccine, still decades away. Fortunately,
there are promising drugs in development or in clinical trial.23
The following drugs are in clinical development: gatifloxacin and
moxifloxacin (phase 2/3), TMC207 (Phase 2), OPC67683 and PA
824 (trials of early bactericidal activity), and SQ109 (Phase 1).
Gatifloxacin and moxifloxacin
Fluoroquinolones are lynchpins for the treatment of MDR-TB.
Gatifloxacin and moxifloxacin have higher in vitro activity and
efficacy in animal models than the other fluoroquinolones, which
has led to interest in their potential use in treatment shortening.
Promising data from a clinical trial in Chennai, India were the
impetus for this approach. Ofloxacin in a multidrug regimen
improved sputum sterilization at 2 months and in 4- and 5-month
treatment regimens was associated with low levels of relapse.24
An important early stage in the evaluation of a new drug is
to assess “early bactericidal activity” or EBA after brief periods
of monotherapy. Moxifloxacin had impressive EBA activity,
greater than rifampin and nearly comparable to isoniazid.25,26 In
published and unpublished studies, moxifloxacin (or gatifloxacin
in one study) substituted for ethambutol accelerated sterilization
of sputum with a similar trend when moxifloxacin was substituted
for isoniazid (Chaisson R, personal communication).27,28 The plan
is to proceed with a Phase 3 trial comparing a 4-month regimen
in which moxifloxacin is substituted for ethambutol (or isoniazid)
with a standard 6-month regimen. This might have some effect on
prevention of XDR-TB, as shorter regimens should be associated
with greater ease of directly observed therapy and generally
improved adherence. The role of moxifloxacin and gatifloxacin in
the treatment of XDR-TB is uncertain, given the cross-resistance
to fluoroquinolones. It also can be argued that more general use of
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fluoroquinolones in drug-sensitive TB may produce higher levels
of drug resistance to this class of agents in patients with MDR-TB,
for whom they have been a mainstay of effective therapy.
Diarylquinoline, R207910 (TMC207)
Perhaps the most promising drug undergoing clinical trials is
diarylquinoline. Johnson and Johnson conducted a drug discovery
program screening lead compounds against Mycobacterium
smegmatis. They rightly claimed that this was the first tuberculosis
(or mycobacteria)—specific drug identified in 40 years. R207910,
the most active compound in the class, showed remarkable
activity against both drug-sensitive and drug-resistant MTB
with a minimal inhibitory concentration of 0.06 µ/µL. This
drug targets the proton pump of adenosine triphosphate (ATP)
synthase. The distinct target precludes cross-resistance with
existing classes of antituberculosis drugs. Further, the activity of
R207910 appeared restricted to mycobacteria, with little activity
against gram positive and gram negative bacteria. This is a useful
property, as the drug is unlikely to be used to treat common
bacterial infections potentially leading to resistance of MTB,
as has occurred with the fluoroquinolones. In fact, early in the
history of antituberculous therapy, it was a dictum that it was
highly desirable that anti-TB drugs be restricted for use against
mycobacteria. There is an extended effect of a single dose of drug
due to long plasma half-life, high tissue penetration, and long
tissue half-life. In mice, R207910 exceeded the bactericidal activity
of isoniazid and rifampin by at least 10 fold. In the mouse model
of established TB infection, R207910 was at least as effective as the
three-drug combination of isoniazid, rifampin, and pyrazinamide.
In summary, the combination of low MIC, distinct mechanism of
action, early and late bactericidal activity, and pharmacokinetic
profile make R207910 an extremely promising drug.
TMC207 has undergone initial Phase 1 studies and is safe
at doses exceeding those that achieved optimal activity in the
mouse model of established infection. In a study of EBA activity
in 75 patients with pulmonary TB, bactericidal activity of TMC207
was observed from day 4 onward and was similar in magnitude to
those of INH and RIF over the same period.28 It is about to undergo
a randomized controlled trial in patients with MDR-TB. There is
an advantage of studying this patient population because of the
larger window to observe an effect compared with adding a new
drug to a combination of active drugs in drug-susceptible TB.
Nitroimidazoles, OPC 67683, and PA 824
A series of bicyclic nitroimidazofurans are agents with potent
in vitro and in vivo activity against MTB.29 Metronidazole, in fact,
showed activity against the dormant stages of MTB.30 The lead
compound in the series is, however, mutagenic. For this reason,
a series of 3-substituted nitro-imidazolpyrans were synthesized.
They showed antituberculous activity but were not mutagenic.31
PA824 was active at submicromolar MICs against drug-susceptible
and drug-resistant isolates of MTB, suggesting a novel mechanism
of action. In mouse and guinea pig models, it is as potent
as isoniazid. PA824 has excellent tissue penetration with levels
3–8 fold higher in lung and spleen than in plasma. Importantly,
P824 is active against dormant as well as replicating forms of MTB.
The mechanism of action of P824 is unknown but it functions
as a pro-drug requiring reductive activation of the aromatic
nitro group that appears to be mediated by a specific glucose-
6-phosphate dehydrogenase or its deazaflavin cofactor.
OPC67683 is a more recently discovered dihydroimidazo-
oxazole. It is remarkably active against MTB in vitro with MICs in
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the range of 0.006 µg/mL. In a mouse model of chronic infection,
it was superior to currently used antituberculous drugs. OPC
67683 is undergoing Phase 1 trials and EBA studies in normal
volunteers.
Pyrrole (LL3858)
The pyrroles were found to have activity against MTB in 1998.32
The mechanism of action of this class of drugs is unknown
but appears to be unique, as activity is similar against drug-
susceptible and drug-resistant isolates of MTB. LL3858 is a
pyrrole with optimized activity, submicromolar MICs and
activity in the mouse model. In combination with current drugs,
LL3858 accelerated the sterilization of infected animals. This
drug is undergoing multidose Phase 1 evaluation in healthy
volunteers in India.
Diamine (SQ109)
This compound was originally intended to be a substitute for
ethambutol and contains its 1,2-diamine pharmacophore.
However, it is dissimilar structurally, appears to have a different
mechanism of action and is quite active in vitro (MIC 0.1–0.63 µg/
mL) and in mice (2–2.5 log reduction in colony forming units in
spleen and lungs).33 The oral bioavailability of the drug is limited
in mice (4%), which may be an issue in clinical use. SQ109 is
undergoing Phase 1 clinical trials in healthy volunteers.
Conclusions
The plethora and diversity of antituberculous drugs in clinical
development is remarkable, given the extended earlier period
without the development of new agents. In fact, it may be the case
that the current first line drugs could be entirely supplanted by these
promising compounds. Ultra-short course treatment and drugs active
against XDR-TB appear within reach. The field has been transformed
not just by governmental grants and contracts, but, more recently,
by the engagement of major pharmaceutical companies—a change
from past disinterest and a very hopeful sign.
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