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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


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


JJ Ellner (jerroldellner@yahoo.com) DOI: 10.1111/j.1752-8062.2008.00086.x 80 Volume 2 • Issue 1 www.ctsJournal.coM

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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


Ellner n The Emergence of Extensively Drug-Resistant Tuberculosis

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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of resistance. Certainly, the high bacterial burden and poor drug penetration of cavities as Volume 2


Ellner n The Emergence of Extensively Drug-Resistant Tuberculosis


The Emergence of Extensively Drug-Resistant Tuberculosis 82 Figure 1. Visual detection of LAMP product under UV
The Emergence of Extensively Drug-Resistant Tuberculosis 82 Figure 1. Visual detection of LAMP product under UV

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. 11 Further, the transfer of 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 Mitchison 14 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 17,18 may relate to increased transmission due to the isonazid resistance mutation, rather than other properties of the Beijing

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

TB vaccines. Most importantly, neither appropriate animals Figure 2. Three-dimensional (3D) reconstruction of M.

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:

Ellner n The Emergence of Extensively Drug-Resistant Tuberculosis

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

MVA85A; Mtb72f; AERAS 402; and Mycobacterium vaccae. MVA85A is a recombinant modified vaccinia virus, expressing

existing classes of antituberculosis drugs. Further, the activity of R207910 appeared restricted to mycobacteria, with little activity

a major secretory product of MTBthe 85A antigen. Mtb72f is

against gram positive and gram negative bacteria. This is a useful

a combination of two immunogenic proteins Mtb 32a and Mtb

39a in two adjuvants ASO2A and ASO1B. It has recently been

property, as the drug is unlikely to be used to treat common bacterial infections potentially leading to resistance of MTB,

reformulated and designated M72. AERAS 402 is a serotype 32


has occurred with the fluoroquinolones. In fact, early in the

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

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

protective vaccines or for use in a prime-boost strategy that will

tissue half-life. In mice, R207910 exceeded the bactericidal activity

include MTB-infected persons.


isoniazid and rifampin by at least 10 fold. In the mouse model


established TB infection, R207910 was at least as effective as the

Development of New Drugs

three-drug combination of isoniazid, rifampin, and pyrazinamide.

New drugs will be necessary to treat the current and projected


summary, the combination of low MIC, distinct mechanism of

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

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

The following drugs are in clinical development: gatifloxacin and


doses exceeding those that achieved optimal activity in the

moxifloxacin (phase 2/3), TMC207 (Phase 2), OPC67683 and PA

mouse model of established infection. In a study of EBA activity

824 (trials of early bactericidal activity), and SQ109 (Phase 1).


75 patients with pulmonary TB, bactericidal activity of TMC207

was observed from day 4 onward and was similar in magnitude to

Gatifloxacin and moxifloxacin

has led to interest in their potential use in treatment shortening.

those of INH and RIF over the same period. 28 It is about to undergo

Fluoroquinolones are lynchpins for the treatment of MDR-TB.


randomized controlled trial in patients with MDR-TB. There is

Gatifloxacin and moxifloxacin have higher in vitro activity and efficacy in animal models than the other fluoroquinolones, which

Promising data from a clinical trial in Chennai, India were the impetus for this approach. Ofloxacin in a multidrug regimen

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

improved sputum sterilization at 2 months and in 4- and 5-month


series of bicyclic nitroimidazofurans are agents with potent

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

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,

of monotherapy. Moxifloxacin had impressive EBA activity,


series of 3-substituted nitro-imidazolpyrans were synthesized.

greater than rifampin and nearly comparable to isoniazid. 25,26 In published and unpublished studies, moxifloxacin (or gatifloxacin

They showed antituberculous activity but were not mutagenic. 31 PA824 was active at submicromolar MICs against drug-susceptible

in one study) substituted for ethambutol accelerated sterilization

and drug-resistant isolates of MTB, suggesting a novel mechanism

of sputum with a similar trend when moxifloxacin was substituted


action. In mouse and guinea pig models, it is as potent

for isoniazid (Chaisson R, personal communication). 27,28 The plan


isoniazid. PA824 has excellent tissue penetration with levels


to proceed with a Phase 3 trial comparing a 4-month regimen

3–8 fold higher in lung and spleen than in plasma. Importantly,

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


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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discovered dihydroimidazo- oxazole. It is remarkably active against MTB in vitro with MICs in Volume 2


Ellner n The Emergence of Extensively Drug-Resistant Tuberculosis

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.


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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