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Keywords:

  • adjunct therapies;
  • drug resistance;
  • extensively drug resistant;
  • genotype;
  • multidrug resistant;
  • Mycobacterium tuberculosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

The first cases of totally drug-resistant (TDR) tuberculosis (TB) were reported in Italy 10 years ago; more recently, cases have also been reported in Iran, India and South Africa. Although there is no consensus on terminology, it is most commonly described as ‘resistance to all first- and second-line drugs used to treat TB’. Mycobacterium tuberculosis (M.tb) acquires drug resistance mutations in a sequential fashion under suboptimal drug pressure due to monotherapy, inadequate dosing, treatment interruptions and drug interactions. The treatment of TDR-TB includes antibiotics with disputed or minimal effectiveness against M.tb, and the fatality rate is high. Comorbidities such as diabetes and infection with human immunodeficiency virus further impact on TB treatment options and survival rates. Several new drug candidates with novel modes of action are under late-stage clinical evaluation (e.g. delamanid, bedaquiline, SQ109 and sutezolid). ‘Repurposed’ antibiotics have also recently been included in the treatment of extensively drug resistant TB. However, because of mutations in M.tb, drugs will not provide a cure for TB in the long term. Adjunct TB therapies, including therapeutic vaccines, vitamin supplementation and/or repurposing of drugs targeting biologically and clinically relevant molecular pathways, may achieve better clinical outcomes in combination with standard chemotherapy. Here, we review broader perspectives of drug resistance in TB and potential adjunct treatment options.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

‘Drug resistance’ is a microbiological diagnosis reflecting the resistance pattern of the clinical isolate of a given pathogen to a defined set of antibiotics. Streptomycin (SM), introduced in 1946 for the treatment of tuberculosis (TB), and SM-resistant M.tb, was first reported in 1947 from the clinical studies with non-responsive patients to this antibiotic [1, 2]. The recognition of this phenomenon, i.e. nonresponsiveness to monotherapy, led to the principle of multiagent chemotherapy for TB, which was proved to be effective in a combination with SM & para-aminosalicylic acid (PAS) [3]. Isoniazid (INH), introduced in 1952, was used alone or in combination with SM or PAS by the British Medical Research Council (BMRC). The first national survey of drug resistance in the UK, organized by the BMRC and led by Wallace Fox, identified Mycobacterium tuberculosis (M.tb) strains resistant to only one of the three available drugs, even prior to treatment, in patients with pulmonary TB [4, 5]. This resulted in the evaluation by John Crofton of a three-drug regimen with SM, PAS and INH, with the notion that two of three drugs would be available for almost any resistant strain [6]. Multi-drug-resistant (MDR) TB was first reported in the 1990s with resistance to INH and rifampicin (RIF), notably with the New York epidemic in human immunodeficiency virus (HIV)-infected individuals [7]. This led to the declaration by the World Health Organization (WHO) of a global TB emergency in 1994. Extensively, drug-resistant TB (XDR-TB) was reported in March 2006 in a joint publication from the Centres for Disease Control and Prevention (CDC) and WHO describing strains of M.tb resistant not only to INH and RIF (i.e. MDR-TB), but also to at least three of the six classes of second-line anti-TB drugs (aminoglycosides, polypeptides, fluoroquinolones, thioamides, cycloserine and PAS) [8, 9]. The determination of drug resistance is dependent on difficult-to-perform drug-susceptibility testing (DST), and some forms of drug resistance are more challenging for diagnosis treatment. Therefore, the definition of XDR-TB was eventually modified at a meeting of the WHO XDR-TB Task Force in October 2006 in Geneva, Switzerland, and is now defined as ‘resistance to at least rifampicin and isoniazid (i.e. the definition of MDR-TB), in addition to any fluoroquinolone, and to at least one of the three following injectable drugs used in anti-TB treatment: capreomycin, kanamycin and amikacin’ [10]. Up to the end of 2012, XDR-TB had been reported in 92 countries (Fig. 1) with an estimated 45 000 new cases per year, that is, 9.6% of all reported cases of MDR-TB [11]. Overall, more than 10 cases of XDR-TB were reported in 13/92 countries in the most recent year for which data are available. The proportion of MDR-TB cases that were classified as XDR-TB was highest in Azerbaijan (Baku: 12.8%), Belarus (11.9%), Latvia (16%), Lithuania (24.8%) and Tajikistan (Dushanbe and Rudaki district: 21%). The proportion of MDR-TB cases resistant to fluoroquinolones and second-line injectable agents was 16.5% and 22.7%, respectively. A total of 32% of patients with MDR-TB exhibited resistance to a fluoroquinolone, to a second-line injectable agent, or to both. Treatment success with second-line therapy is only achieved in 12–28% of XDR-TB cases, as reported in the latest WHO Global TB Report, with mortality as high as 44–49%. Globally, only 1557 patients with XDR-TB received adequate second-line therapy in 2012. Thus, urgent action is required to close the XDR-TB treatment access gap, and to consider new treatment options.

image

Figure 1. Global map of drug-resistant tuberculosis (TB). Countries with at least one reported case of extensively drug-resistant TB are shown in pink; four countries with reported cases of totally drug-resistant TB (Italy, Iran, India and South Africa) are also shown (in dark pink). Adapted from [11].

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The prevalence of MDR-TB in a country correlates with the duration of availability of second-line treatment. In countries in which second-line TB treatment has been available for more than 20 years, higher rates of resistance have been observed compared with those with a shorter period of treatment availability [12].

Totally drug-resistant tuberculosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

Totally drug-resistant tuberculosis (TDR-TB) refers to M.tb clinical strains that show in vitro resistance to all first- and second-line drugs tested (INH, RIF, SM, ethambutol, pyrazinamide, ethionamide, PAS, cycloserine, ofloxacin, amikacin, ciprofloxacin, capreomycin and kanamycin). The presence of TDR-TB was first observed in two patients in Italy in 2003 and subsequently reported by Migliori et al. in 2007 [13]. These M.tb strains were also resistant to rifabutin, clofazimine, dapsone, clarithromycin and thiacetazone. The authors suggested the new term ‘XXDR-TB’ to define this (TDR-TB) strain. The second report of TDR-TB came from Iran: 10.3% of clinical M.tb isolates from 146 patients with MDR-TB were in fact found to be TDR-TB [14]. According to the WHO TB Report 2013 quoting results from 2010, treatment success rate was 48% for MDR-TB and 22–28% for XDR-TB, with a mortality rate of 44–49% in patients with XDR-TB [11].

In January 2012, there was a report of four cases of TDR-TB in India (one of the countries with the highest burden of drug-resistant TB) with resistance against INH, RIF, SM, ethambutol, pyrazinamide, ethionamide, PAS, ofloxacin, amikacin, ciprofloxacin, capreomycin and kanamycin (cycloserine was not tested). The situation caused widespread media attention leading to a consultative expert group meeting at WHO [15]. The WHO consultative meeting on TDR-TB did not officially endorse the ‘TDR-TB’ terminology, which still remains to be fully defined (e.g. resistance to the entire spectrum of anti-TB drugs, including drugs that may be under development). Recently, three new drugs, bedaquiline, delamanid and linezolid have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency that may offer therapeutic solutions for TDR-TB. With more new anti-TB agents in the pipeline, there is hope of identifying drugs that may be bactericidal or bacteriostatic in TB treatment and thereby challenging the TDR-TB terminology.

Three of the four patients from India with TDR-TB died as a result of their disease, despite treatment with double-dose INH, linezolid, clofazimine, thioridazine, meropenem and clavulanate. In a recent update on TDR-TB, 15 cases with resistance to all drugs tested (as above) have been reported, and five patients died due to their disease; salvage regimens included surgical resection of pulmonary lesions [16].

Using transmission and atomic force microscopy, morphological variation in TDR-TB M.tb isolates was identified, with round, oval and multiple branching bacillary forms with thicker cell walls and various types of symmetrical/asymmetrical budding formations during the exponential growth phase [17-19]. However, the impact of structural differences in M.tb on transmissibility and/or the quality and strength of immune responses remains unclear.

Overall, 92% of 236 MDR M.tb strains collected in South Africa (Stellenbosch University) belonged to an atypical Beijing genotype resistant to 10 anti-TB drugs (four first line: INH, RIF, pyrazinamide, ethambutol and six second line: SM, amikacin, kanamycin, capreomycin, ethionamide and ofloxacin). Some of these M.tb strains were also resistant to PAS, indicating emergence of TDR-TB in South Africa [20]. In total, TDR-TB has only been reported from four countries (Fig. 1). This may under-represent the real extent of drug resistance in M.tb isolates because of many factors, and in particular, constrained laboratory access in resource-limited settings with a high prevalence of TB.

Clinical Mycobacterium tuberculosis spread and diagnosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

To date, more than 30 cases of TDR-TB have been reported; a majority of these cases succumbed to the disease. This is likely to be a very small number of the total cases, as drug-resistant TB may be undetected due to the lack of diagnostic capacity. Ninety-two countries reported cases of XDR-TB. However, in Africa (the continent with the highest TB rates per capita and including the country – South Africa – with the most reported cases of XDR-TB), only 17 out of 54 countries reported cases of XDR-TB [11]. It was reported that two of the 27 countries worldwide with the highest burden of MDR-TB routinely tested for second-line drug resistance [21]. Lack of capacity and inconsistent methods for drug resistance testing remain some of the biggest challenges to understanding the prevalence of TDR-TB.

Lack of sufficient capability and capacity to test for drug resistance in M.tb isolates are not the only challenges in diagnostics; the lack of ability to test new therapeutic options is also a concern. It is possible that antibiotic combination therapy may be effective in vivo even though there is resistance to individual drugs in vitro. Clinical response to antibiotic combinations may also be influenced by inter-individual differences in pharmacodynamics as well as by the genetic make-up of the individual patient. Therapeutic drug monitoring has been proposed to evaluate desired drug concentrations and the dynamics of resistance patterns in patients with XDR-TB or TDR-TB [22].

Standard methods for DST employ M.tb culture on solid media (8–12 weeks) and in liquid media (2–4 weeks), both of which take a long time before results are obtained during which inappropriate drugs might be administered with increased resistance profiles as a result. Faster liquid media systems including the Mycobacteria Growth Indicator Tube (MGIT) system provide faster results, yet costs and infrastructure requirements limit their clinical use in many parts of the world. Genotype-based tools provide the fastest way to diagnose resistance mutations in M.tb, yet genetic analysis requires further characterization of M.tb with DST (phenotypical). The Xpert MTB/RIF is approved by the WHO to provide a relatively fast diagnosis of TB as well as a test of RIF resistance [23]. Genotype MTBDRplus is a second-generation line-probe assay (LPA) for rapid diagnosis of resistance to RIF and INH in culture medium [24]. Genotype MTBDRsl is the advanced version of LPA to detect XDR-TB (resistance to fluoroquinolones, aminoglycosides/cyclic peptides and ethambutol) in already characterized MDR M.tb isolates [25].

Other methods include, for example, use of TK medium [26], the microscopic observation drug-susceptibility assay (MODS) [27] and the FASTPlaque-Response bacteriophage assay [28]; molecular methods are based either on nucleic acid amplification combined with electrophoresis, hybridization and sequencing, direct sequencing, reverse hybridization [29] or a TB Biochip platform [30].

Causes of TB resistance

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

Factors affecting the mutation rate of bacteria can be divided into two groups: (i) cellular mechanisms, such as lack of mismatch repair, microsatellites, existence of drug resistance-conferring mutations, mistranslations and error-prone DNA polymerases and (ii) external factors, such as antibiotics, anti-retroviral drugs, host environment and smoking (reviewed in detail in [31]). There are two types of drug resistances: primary (transmitted) and acquired. The development of acquired MDR-/XDR-/TDR-TB is a multifactorial process. Treatment of TB disease depends on the health care system (e.g. DST, antibiotic medicines, control programmes, access to medical facilities and quality of drugs), the treating doctor (e.g. type of drug, dose of drug, drug interactions and duration of treatment) and the patient (e.g. adherence to therapy, individual pharmacokinetics [32] and co-infections). Also the use of anti-TB drugs to treat other bacterial infections in patients who are positive for M.tb might contribute to the emerging resistance [33]. Primary drug-resistant TB occurs due to direct transmission of the drug-resistant strains of M.tb resulting in primary TB disease. This has been well established through molecular epidemiology and contact tracing, confirming transmission of M.tb strains.

It seems that most patients who acquire XDR-/TDR-TB have been previously treated with anti-TB drug. Despite successful completion of the previous treatment, a small fraction of the bacilli might survive as ‘persisters’ and acquire drug resistance following withdrawal of the drug-induced pressure, before ‘growing out’ and causing more resistant forms of the disease. This often occurs in a sequential way in which increasingly more resistance is acquired over time when treatment with second-line drugs is initiated. Indeed, previous treatment with second-line anti-TB agents, if not properly managed, is one of the strongest risk factors for acquiring XDR-TB [12].

Examples of acquired antibiotic resistance mechanisms include passive resistance, modification of drug targets, chemical drugs modification, enzymatic drug degradation, molecular mimicry of drug targets, drug deportation by efflux pumps and epigenetic drug tolerance [34]. Acquired antibiotic resistance mechanisms often result in reduction in fitness of the resistant M.tb mutants, which in turn determine the stability and further propagation of the resistance phenotype. However, there can be compensatory mutations that restore the fitness of the resistant strains, stabilizing the emerging resistance phenotype and thereafter enhancing transmission amongst common populations [35, 36].

The microenvironment at the site of TB disease is influenced by many factors, such as the immune response profile, severity of inflammation and cellular necrosis, and oxygen availability. These factors play a crucial role in M.tb mutation rates and selection of resistant M.tb strains. Jenkins et al. showed an increased M.tb mutation rate at low pH as a result of M.tb adaptation to stress causing upregulation of error-prone polymerases [37]. M.tb isolates from the cavity wall, pericavity tissue or apparently healthy tissue demonstrated different transcriptional profiles. Other environment-related, non-genetic mechanism of drug resistance are transcriptional changes, such as transcriptional activity of drug efflux pumps in M.tb (reviewed in [38]).

Mycobacterium tuberculosis genotype and drug resistance

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

Virulence, immunogenicity and drug resistance all vary between different M.tb strains. Bacterial chromosomal mutations result in drug resistance against anti-TB drugs. These mutations are rare and random events. The average M.tb mutation rates for INH, SM, ethambutol and RIF are 2.56 × 10−8, 2.95 × 10−8, 1.0 × 10−7 and 2.25 × 10−10 mutations per bacterium per generation, respectively [39]. The highest proportion of mutants that can be expected in an unselected bacterial population was found to be 3.5 × 10−6 for INH and 3.1 × 10−8 for RIF. A double-resistant M.tb mutant is therefore estimated in a population of 1015 bacilli. A recent mathematical model addressed the issue of spontaneous emergence of MDR-TB taking into account the dynamic interplay between innate and acquired immune responses of the host leading to death of certain M.tb populations. The probability of the development of drug resistance during clonal expansion of an initially sensitive TB infection is the orders of magnitude higher than previously estimated [35]; the probability of MDR, at the time of diagnosis, may be 1000–10 000 times higher than previously considered. Caution may be required as dual-resistant M.tb strains are expected in a small minority of patients even prior to treatment; standard drug regimens administered properly are likely to result in the selection of the preexisting dual resistance. M.tb mutations and TB drug resistance data can be retrieved from the TB drug resistance mutation database (http://www.tbdreamdb.com/) [40].

Whole-genome sequencing to define the genetic determinants of drug resistance

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

There have been a number of international attempts across many institutions in different geographical regions to identify the entire repertoire of naturally-occurring mutations in M.tb responsible for drug-resistant TB to create a catalogue of resistance-conferring mutations by sequencing large numbers of geographically and phenotypically diverse M.tb strains. These M.tb strains have been characterized according to their resistance to a broad spectrum of first- and second-line antibiotics. The aim of these efforts has been to: (i) guide development of rapid tests for accurate diagnosis of drug resistance in TB, (ii) guide TB treatment and (iii) discover new targets for developing improved anti-TB therapeutics.

Leading institutions involved in this endeavour include the Broad Institute (http://www.broadinstitute.org/annotation/genome/mycobacterium_tuberculosis_diversity/MultiHome.html, http://www.broadinstitute.org/science/projects/gscid/genomic-sequencing-center-infectious-diseases); the Sanger Centre, UK (http://www.sanger.ac.uk/resources/downloads/bacteria/mycobacterium.html); the Swiss Tropical and Public Health Institute (http://www.swisstph.ch/about-us/departments/medical-parasitology-infection-biology/tuberculosis-research.html); the University of California, San Francisco [41]; the German Research Centre, the Leibniz Centre of Medicine and the Biomedical Research, Borstel, Germany [42, 43] and the Institute of Biophysics, the Chinese Academy of Sciences, Beijing, China [44]. This list is not comprehensive, but reflects the international effort to better map and understand M.tb diversity.

Whole-genome sequencing (GWS) approach for rapid high-resolution M.tb genotyping is an attractive emerging tool for the determination of the broad range of genes involved in the generation of XDR-/TDR-TB. A recent study using a GWS approach confirmed all 11 drug resistance markers known to date. In addition, 39 new genes associated with resistance were described. It is interesting to note that most of the newly reported mutations were found to be associated with M.tb cell wall permeability. This information is vital because drug-resistant M.tb strains may contain mutations not only in genes coding for key enzymes, but also in those coding for cell wall remodelling [45]. M.tb genes and related enzymes targeted by drugs and/or associated with drug resistance are listed in the Table 1 [128].

Table 1. Mutations associated with antibiotic resistance in Mycobacterium tuberculosis
AntibioticsGenes involved in drug resistance (and their proteins)
  1. Adapted from Köser et al. (Ref. 128) Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis.

Amikacin, capreomycin, kanamycin, streptomycin and viomycinrpsL (Rv0682), tap (Rv1258c), rrs (MTB000019), rrl (MTB000020), tlyA (Rv1694), eis (Rv2416c), whiB7 (Rv3197A) and gid (Rv3919c)
SQ109mmpL3 (Rv0206c)
AZD5847, Linezolid and sutezolidrplC (Rv0701) and rrl (MTB000020)
BedaquilineatpE (Rv1305)
ClarithromycinermMT (Rv1988) and whiB7 (Rv3197A)
Clofazimine and thioridazinendh (Rv1854c)
CycloserinecycA (Rv1704c), ddlA (Rv2981c) and alr (Rv3423c)
Delamanid and PA-824fgd1 (Rv0407), fbiC (Rv1173), fbiA (Rv3261), fbiB (Rv3262) and ddn (Rv3547)
EthambutolembR (Rv1267c), embC (Rv3793), embA (Rv3794) and embB (Rv3795)
Gatifloxacin, levofloxacin, moxifloxacin and ofloxacingyrB (Rv0005) and gyrA (Rv0006)
Imipenem, meropenem and co-amoxiclavpbpA (Rv0016c), blaC (Rv2068c), ldtA (Rv0116c), ldtB (Rv2518c) and dacB2 (Rv2911)
Isoniazid, prothionamide and thiacetazonemshA (Rv0486), hadA (Rv0635), hadB (Rv0636), hadC (Rv0637), mmaA4 (Rv0642c), mmaA3 (Rv0643c), mmaA2 (Rv0644c), mshB (Rv1170), sigI (Rv1189), fabG1 (Rv1483), inhA (Rv1484), ndh (Rv1854c), katG (Rv1908c), furA (Rv1909c), mshC (Rv2130), ahpC (Rv2428), nudC (Rv3199c), nat (Rv3566c), ethA (Rv3854c) and ethR (Rv3855)
Para-aminosalicylic acidand co-trimoxazolefolC (Rv2447c), ribD (Rv2671), dfrA (Rv2763c), thyA (Rv2764c) and folP1 (Rv3608c)
PyrazinamiderpsA (Rv1630), pncA (Rv2043c) and panD (Rv3601c)
Rifampicin, rifapentine and rifabutinrpoB (Rv0667), rpoC (Rv0668) and rpoA (Rv3457c)

Clinical in vitro nonresponsiveness

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

Phenotype and drug resistance

In the clinical practice of drug-resistant TB management, patients often demonstrate early culture conversion to negativity during the first 3 months after initiation of second-line therapy with reversion to culture positivity several months later. Also, in many instances, patients may not exhibit clinical improvement, despite appropriate second-line therapy in conjunction with the in vitro DST results. The issues of re-infection or ‘super-infection’ acquired in most cases in the hospital setting and of diseases with multiple drug-resistant M.tb strains versus microbiological testing complicate the clinical reality of patient management.

Phenotype versus genotype

Phenotypic analysis of DST is essential to corroborate the genotypic detection of drug-resistant TB to determine the optimal therapeutic regimen for each patient. This can be performed either by automated liquid culture (MGIT system) or by solid culture methods. Other cost-effective options include MODS, slide DST, phage-based assays, colorimetric methods with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) and resazurin as redox indicators and the nitrate reductase assay. It is also essential to corroborate genotypic diagnosis with phenotypic characterization to follow up patients on therapy. In recent years, there have been several reports of deterioration of patients despite specific therapy following DST, suggesting a drift in the drug resistance patterns with modification of phenotypic characteristics in terms of susceptibility and resistance [46]. Hence, a proportional concentration method with multiple antibiotic concentrations is recommended rather than using a single critical concentration. This is particularly important for patients who deteriorate following therapy.

In a recent review, it was elegantly emphasized that the term ‘resistance’ with regard to M.tb is by no means a simple homogeneous category; instead it represents several heterogeneous factors frequently composed of low-, moderate- and high-level drug resistance [47]. Low-level drug resistance may not necessarily correspond to clinical drug resistance; conversely, in the presence of a high-level resistance phenotype, the drug may be of little, if any, clinical benefit. The clinical implications of moderate levels of resistance are less clear, and need to be addressed in prospective studies with standardized protocols for quantitative DST of both first- and second-line therapy for assessing the impact of resistance heterogeneity on treatment results.

Host versus drug-resistant Mycobacterium tuberculosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

The biology and epidemiology of human TB depends on a delicate balance between host (e.g. genetic variants of factors in the immune system) and pathogen-derived factors (e.g. virulence factors and escape mutations) as well as co-infections (e.g. HIV, malaria and helminth infections), comorbidities (e.g. malnutrition and stress) and other associated host factors such as poverty. Different M.tb strains display a geographically structured transmission pattern, suggesting adaptation between different human populations and M.tb strains [36, 48]. Individuals infected with more ‘modern’ M.tb lineages are more prone to develop active TB. This is also associated with decreased early inflammatory responses as compared with individuals infected with more ‘ancient’ M.tb lineages [49]. The more ancient M.tb lineages have probably been circulating in the human population for a longer time and immune responses might have adapted to these pathogenic variants. This ‘co-evolution’ of the pathogen–host relationship needs to be further examined. The effect of chemotherapy in the context of adaption to M.tb strains remains to be investigated, yet some strains have shown a tendency towards developing drug resistance mutations such as the Beijing W genotype [50]. Although genetic differences, including insertions, deletions and point-mutations, between different M.tb strains are believed to be low [51], they may result in phenotypic changes affecting the virulence and infectiousness of the strains [52]. Most of the genetic variation resides in nonessential genes and in parts of the M.tb genome that are not believed to be presented to the human immune system [53]. This has led to the hypothesis that immune recognition may be beneficial for the pathogen and not for the host; however, this notion requires further testing.

A high degree of phenotypic variation between M.tb clinical isolates has been found to affect both the spread of the specific strain and the clinical outcome of infection. M.tb strains derived from the East-Asian lineage are able to acquire resistance more rapidly as compared with the Euro-American lineage. However, in the East-Asian lineage, Ford et al. found no association between the increased ability to generate drug resistance and the enhanced ability to survive and mutate in the presence of drug(s), and no evidence of a ‘fitness cost’ of such mutations [54]. Notably, in a previous study [using the genome-wide association study (GWAS) approach], the same authors found that M.tb could acquire mutations even in the latent state [55].

The M.tb Beijing genotype is associated with ‘hypervirulence’ as well as with an MDR phenotype [50]. A different, commonly circulating M.tb genotype, Haarlem, has been shown to be over-represented in patients with drug-resistant TB [56]. The fact that certain strains are more prone to acquiring drug resistance is of course of particular interest in the context of TDR-TB: 10 out of 16 patients with TDR-TB (in Iran) were infected with either Haarlem or Beijing superfamily of strains [14]. The Beijing genotype seems to be able to retain fitness despite the acquisition of drug resistance [57]; this is a very dangerous quality in the context of the drug-resistant TB epidemic. Another phenotypic difference between certain susceptible and MDR-TB strains is the capacity to stimulate immune cells to produce IL-17. This is thought to be due to differences in the bacterial cell wall of the strain, affecting their recognition by pattern recognition receptors of the innate immune system [58].

In the context of mutations in the TDR-TB genome, the problem is not exclusively the mutations that induce resistance, but also those acquired by a strain that is situated in a part of the TB genome presented to the immune system. Mutations may cause immune escape mechanisms, and such mutations in T cell epitopes might affect the efficacy of therapeutic TB vaccines (Fig. 2).

image

Figure 2. Factors that affect totally drug-resistant tuberculosis (TDR-TB). Interaction and adaptation between the host and the mycobacterium are well documented. The host immune system drives Mycobacterium tuberculosis (M.tb) towards genetic and phenotypic changes, often increasing virulence. Factors (inflammation, necrosis and oxygen availability) within the microenvironment (e.g. the lung) affect the mutation rate of M.tb as well as the transcriptional profile and thereby genotype and phenotype. The bacterial phenotype might influence the host response by exhausting immune responses and introducing anergy in immune cells targeting the pathogen. Bacterial escape mutations might also disarm the immune response. External factors, such as anti-TB drugs, might influence both the genotype and phenotype by introducing drug resistance mutations in the bacterial genome which will give rise to a multidrug-resistant phenotype. The host environment affects the absorption and pharmacokinetics of the drugs; other medications might also influence the effect of anti-TB drugs. Drug resistance mutations might also arise in M.tb epitopes leading to immune escape mutations. Other ways of targeting the TDR bacteria might be through the use of therapeutic vaccines with the aim of an improved and more focused immune response through change from a nonproductive Th2 to a Th1 response; this may also involve the reduction in nonproductive inflammation in TB. MSC, mesenchymal stromal cells; VitD, vitamin D.

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TDR-TB, co-infection and comorbidity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

Many factors have been associated with increased risk of acquiring active TB or worsening the outcome of TB infection, including HIV infection [59], diabetes mellitus [60, 61], renal disease [62], solid organ transplantation [63], tumour necrosis factor alpha (TNFalpha) antagonist treatment [64], cancer [65], alcohol abuse [66] and tobacco use [67]. Only a few of these factors, for example, diabetes mellitus [68], have also been associated with higher risk of developing MDR-TB whereas others, such as HIV infection and alcohol abuse [69], have been associated with a worse outcome of MDR- or XDR-TB. Despite diabetes mellitus being a risk factor for developing MDR-TB, TB disease outcome does not seem to be worse amongst diabetic patients [70]. However, the opposite appears to be true for HIV infection. To date, it has not been conclusively proven that HIV infection leads to a higher probability of developing MDR-TB disease [71] (only an increased risk of acquired RIF resistance), but HIV has been associated with numerous outbreaks of MDR-TB. Co-infection with MDR- or XDR-TB is life-threatening to individuals infected with HIV [9, 72]. In a study conducted in South Africa, the mortality rate was as high as 98% in patients co-infected with XDR-TB and HIV [9], and a mortality rate of 80% in co-infected individuals in New York (compared with 47% in individuals only infected with MDR-TB) was also reported [7]. Another complication of HIV–TB co-infection is that HIV is associated with gastrointestinal malabsorption of anti-TB drugs which might lead to acquired resistance [73]. Furthermore, interactions between anti-retroviral and anti-TB drugs lead to limited treatment options and thereby complicate the treatment of drug-resistant TB further in co-infected patients.

Because only a limited number of cases of TDR-TB have been reported, it is currently too early to determine the influence of comorbidities. The Iranian TDR-TB patients were all HIV negative [14], and information regarding comorbidities was lacking for the Italian and Indian TDR-TB cases [15, 16]. Clinical information was not available in the South African study, thus the HIV status of these patients was uncertain; however, as the general HIV prevalence in South Africa amongst TB patients is high, it is likely that at least some were infected with HIV.

Novel anti-TB drugs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

The desired attributes of a novel anti-TB drug are: (i) a new mode of action; (ii) good tolerability; (iii) good pharmacokinetic profile and bioavailability; and (iv) no interactions with other TB or HIV drugs. By implication, a drug with these attributes could be used against MDR-/XDR-/TDR-TB in adults (as well as in children) together with anti-viral therapy against HIV. There are different types of novel anti-TB drugs undergoing clinical trials, both ‘repurposed’ drugs and novel chemical compounds that belong to already existing drug classes or classes with a completely new mode of action.

Delamanid (OPC-67683) and PA-824 are compounds that belong to the nitroimidazole antibiotics, which have been used since the 1950s to treat anaerobic bacterial and parasitic infections [74]. Delamanid and PA-824 are pro-drugs and are activated by M.tb enzymes. The effect of these drugs is thought to be different under aerobic and anaerobic conditions. Under anaerobic conditions, the drugs generate nitric oxide (NO) leading to NO poisoning of the cytochrome C oxidase [75]. Under aerobic conditions, they block the synthesis of mycolic acid, leading to destabilization of the mycobacterial cell wall [76]. Both delamanid and PA-824 are active against replicating and nonreplicating bacteria and are currently in late-stage clinical trials for the treatment of MDR-TB. In a recent study published by Gler et al. in the New England Journal of Medicine, it was shown that delamanid could increase sputum conversion in MDR-TB patients [77]. Another next generation nitroimidazole with more potent anti-mycobacterial properties is TBA-354. This compound is currently undergoing preclinical toxicity testing.

Bedaquiline (TMC207), which belongs to the diarylquinoline antibiotics, blocks ATP synthase leading to lower intracellular ATP levels [78]. This drug candidate exhibits a long half-life and is potent against both replicating and dormant M.tb [79]. It has been tested in Phase II clinical trials in combination with standard treatment for MDR-TB, and demonstrated shorter culture conversion time and an increased proportion of patients achieving culture conversion [80]. It has received fast-track approval from the FDA for use in patients with MDR- and XDR-TB [81], however, it carries a warning due to an association with fatal arrhythmia.

The ethylenediamine SQ109 was originally synthesized as an analogue of ethambutol but with completely different mode of action; it is therefore active against many ethambutol-resistant strains [82]. SQ109 inhibits mycobacterial growth by inhibiting the assembly of mycolic acid into the mycobacterial cell wall [83]. It has the same high minimum inhibitory concentration for both drug-susceptible and drug-resistant M.tb and no antagonistic drug interactions with first- and second-line drugs have been detected [84]. On the other hand, it has been shown that SQ109 has synergistic effects with bedaquiline because it weakens the cell wall of the mycobacterium, thereby allowing for bedaquiline to reach its target ATP synthase more easily [85]. In addition, synergistic effects of SQ109 with INH and RIF have been reported [86]. Another interesting characteristic of this drug is its very low mutation rate (2.55 × 10−11 mutations per bacterium per generation), which gives it a desirable profile for inclusion in treatment of patients with relapsing/recurrent TB. SQ109 is currently in Phase II clinical trials for drug-sensitive TB [84].

A new class of antibiotics that was recently discovered is the oxazolidinones [e.g. linezolid, sutezolid (PNU-100480) and AZD5847]; the novel mechanism of action of this group is inhibition of protein synthesis by blocking parts of the ribosome [87]. No cross-reactivity with other anti-TB drugs has been reported. Linezolid was first used for treating antibiotic-resistant Gram-positive bacteria due to its broad spectrum of activity, but it has recently been evaluated for the treatment of MDR- and XDR-TB [88, 89]. The disadvantage of linezolid is its side effects, including neuropathy and myelosuppression [88]. Sutezolid is an analogue of linezolid with a superior ability to kill M.tb along with improved safety profile [90]. It is a novel compound currently in Phase II clinical trials in patients with MDR-/XDR-TB, and has demonstrated a lower minimum inhibitory concentration than linezolid towards clinical isolates of MDR-TB [91]. Furthermore, AZD5847 has been shown to have superior efficacy and an improved safety profile [92] and is currently in Phase II clinical trials (Table 2).

Table 2. Novel drugs for the treatment of tuberculosis
Novel drugsClinical phaseDrug classMode of action
DelamanidIIINitroimidazoleGenerate NO, block synthesis of mycolic acid, destabilize cell membrane
PA-824IINitroimidazoleGenerate NO, block synthesis of mycolic acid, destabilize cell membrane
BedaquilineIIDiarylquinolinesInhibit ATP synthase, decreasing intracellular ATP level
LinezolidIIOxazolidinonesBlock the 23S rRNA, inhibiting protein synthesis
SutezolidIIOxazolidinonesBlock the 23S rRNA, inhibiting protein synthesis
AZD5847IIOxazolidinonesBlock the 23S rRNA, inhibiting protein synthesis
SQ109II1,2-diamineInhibit the mycolic assay transporter MmpL3, inhibiting assembly of the cell wall

Other medicines that have been repurposed as anti-TB drugs in the context of MDR-TB include the recently developed fluoroquinolones moxifloxacin and gatifloxacin, as well as the anti-leprosy drug clofazimine [93].

Traditionally, drug development progresses through target-based approaches, which unfortunately have not yielded many viable end products in the case of anti-TB drugs. Hence, recent approaches using phenotypic anti-bacterial whole-cell assays have helped to drive the newer compounds (such as SQ109, PA-824 and bedaquiline) in the current drug development pipeline.

Multidrug regimens are essential for avoiding resistance. Each drug in a regimen can have a synergistic or antagonistic effect on the bioavailability of the others. This makes it difficult to extrapolate efficacy and safety data between different regimens and call for extended testing of different regimens and different dosing prolonging the need for extensive and complicated clinical trials. New drugs such as sutezolid, delamanid, bedaquiline, PA-824 and linezolid may provide hope for effective treatment of patients with TDR-TB. Susceptibility to these drugs in the so-called TDR-TB strains needs to be determined. Novel drugs can be used together with already established regimens or as a completely new treatment. Therefore, SQ109 and sutezolid combined [94] or individually sutezolid, bedaquiline, PA-824, SQ109 and pyrazinamide [95] hold promise as potential treatment options for XDR-TB.

The discovery of a new ATPase-inhibiting anti-TB drug candidate, Q203, which acts on the QcrB subunit of the M.tb cytochrome bc1 complex, has recently been reported [96]. Q203 demonstrates high potency in vitro and in vivo against intracellular drug-susceptible M.tb (H37Rv) as well as MDR-TB and XDR-TB clinical isolates. Additionally, Q203 has an impressive safety profile; it enhances bactericidal effects [~3.5-log reductions in lung colony forming units (CFU)] with minimal pathology (dampened alveolar inflammation) in a mouse model of TB. Q203 is currently under preclinical evaluation. Because new anti-TB drugs will be used as part of combinatorial therapy, the issue of drug–drug interactions has been addressed by assessing the possible inhibition by Q203 of the human pregnane X receptor, cytochrome P450 and the P-glycoprotein efflux transporter. No drug–drug interactions have been found, which, in addition to the absence of cardiotoxicity and the high pulmonary bioavailability, add to the favourable profile of Q203.

Adjunct treatments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

Therapeutic vaccines

The development of novel anti-TB drugs, drug regimens and other adjunct therapies will temporally help in the fight against the totally resistant strains of M.tb, but even if the new treatment options are carefully implemented, sooner or later M.tb will once again acquire resistance. Therefore, the only long-lasting solution to fight drug-resistant TB may be to develop improved vaccines against the disease. Vaccines could be either prophylactic or therapeutic; the development of postexposure vaccines or cell-based immunotherapies might complement the currently used chemotherapy, including new anti-TB drugs. The purpose of adjunct immunotherapies is to engage the immune system of the infected individual to provide help in controlling the disease, either by directing the immune system to a productive immune protective response or by targeting persistent bacteria, that current chemotherapies are not able to eradicate. This might lead to shortened regimens of drug treatment, better compliance rates, higher cure rates, significant cost-savings and, hopefully, lower transmission rates. These issues, especially increased compliance, are particularly important in preventing the occurrence of relapses as well as MDR-, XDR- and TDR-TB. Adjunct immunotherapy might also provide a new option for patients with, otherwise, limited treatment choices, such as those with extensive pulmonary damage. Even if adjunct therapies lead to only a transient effect, they could still be of value by slowing down TB disease to provide time for DST and alternative therapeutic interventions.

Several therapeutic TB vaccine candidates are currently under development. Unfortunately, none is directed specifically at, or being tested in the context of, MDR-/XDR-/TDR-TB. TB vaccines that have reached clinical trials comprise either inactivated forms of M.tb, that is, RUTI [97] or mycobacteria other than TB, such as Mycobacterium indicus pranii [98] or Mycobacteria vaccae [99]. The vaccine based on M. vaccae consists of heat-killed bacteria and has been shown to promote an immune Th1 response [100], whereas the RUTI vaccine consists of detoxified fragments of antigens associated with M.tb latency. Immune responses elicited by RUTI might therefore target persistent mycobacteria. It has been proposed that combining these two therapeutic vaccines may be effective due to their different modes of action [101]. ID93/GLA-SE and H56 are two other therapeutic vaccine candidates (both contain four M.tb antigens associated with virulence or latency) in preclinical testing, which at least in animal models resulted in an improvement in survival and reduced pathology and bacterial load in the lungs [102, 103]. In future, it might also be interesting to investigate the potential use of other vaccine candidates that are currently undergoing trials (e.g. VPM1002 [104] and HBHA), in the context of alternative use as therapeutic vaccines for treating drug-resistant forms of M.tb.

Cell-based immunotherapies

Cell-based immunotherapies might also be interesting as adjunct therapies in the context of TDR forms of M.tb. Following suggestions that they may provide an attractive immunotherapeutic option for immunocompromised individuals with MDR-TB [105], dendritic cell-based vaccines (either gene modified or subunit) are being tested at present in a mouse model. In addition, macrophages infected with M.tb have been tested in a mouse model of TB [106]. In a recent study using autologous mesenchymal stromal cells in patients (n = 30) with MDR-/XDR-TB, with the aim of reducing nonproductive pulmonary inflammation, restoring M.tb-directed cellular immune responses and restoring pulmonary function by tissue repair/regeneration, it was found that this treatment was safe and well tolerated [107]. Similar approaches may be effective for adjunct treatment of patients with TDR-TB.

Vitamin supplementation

Nutritional status is known to affect the risk of acquiring active TB. Vitamin D has been shown to influence TB-directed immune responses [108]. In addition, vitamin D and its metabolites have been shown to have an effect on monocytes/macrophages. Because the infection of macrophages is a key step in M.tb pathogenesis, vitamin D supplementation has received attention as an adjunct treatment for TB. It has been reported that vitamin D supplementation accelerates the resolution of adverse inflammatory responses [109] by suppressing proinflammatory cytokine responses [110] as well as by influencing epigenetics [111]. The metabolically active vitamin D metabolite 1,25-dihydroxyvitamin D3 (calcitriol), which is formed by hydrolysis of circulating calcifediol, modulates adaptive immune responses by suppressing interferon-γ and IL-2 production of Th1 cells as well as 1L-12 production and major histocompatibility complex II expression of antigen-presenting cells [112]. However, the vitamin D might also act directly on M.tb. Calcitriol has been shown to have an anti-mycobacterial effect by inducing: (i) reactive oxygen and nitrogen intermediates; (ii) the transcription of anti-microbial peptides such as cathelicidin and (iii) autophagy. Cathelicidin is induced by the binding of vitamin D and the vitamin D receptor to its promoter [113]. A relationship has been shown between the anti-microbial activity of macrophages and cathelicidin expression [114]. The peptide might have dual effects in cells by (i) inducing autophagy, that is, degradation of cytoplasmic components via the lysosomal pathway, which may offer protection against infection with M.tb [115, 116] and (ii) increasing the efficiency of macrophages to kill mycobacteria [114].

Several studies of vitamin D supplementation in TB patients have been conducted with mixed results based on clinical outcomes [109, 117, 118]. It might be interesting to pursue this approach in the context of MDR-TB infections as it has been shown that adjunct therapy with vitamin D is effective to some degree in reducing immunopathological inflammation [109], which is associated with IL-17 production and cell wall composition of certain MDR strains of M.tb [58].

Another vitamin with potential bactericidal effect on M.tb is vitamin C. High doses of vitamin C killed MDR- and TDR-TB strains in vitro; the mycobacteria did not develop any resistance to this treatment [119].

Repurposing of drugs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

Repurposing of drugs, such as the recently reported anti-tubercular activity of ibuprofen [120] and verapamil [121, 122], highlights the possibility of expanding the spectrum of currently available chemotherapeutic agents against TB. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) potentially present a novel class of antibiotics for future consideration. PPMOs are chemically stabilized nucleic acids that bind to bacterial mRNA, consequently silencing gene expression, and they have been shown to exert gene-specific anti-bacterial effects against both enteropathogenic Escherichia coli and Salmonella typhimurium [123]. Pathogen specificity and low toxicity prompted the evaluation of PPMOs in the context of hospital-acquired bacterial infections. Potent in vitro and in vivo bactericidal activity of PPMOs against MDR strains of the pulmonary pathogens Burkholderia multivorans [124] and Acinetobacter baumannii [125] was successfully shown. Encouraging results have also been reported with PPMOs against tachyzoites of the apicomplexan parasite Toxoplasma gondii, which, like M.tb, represents a persistent intracellular pathogen [126]. As such, the results of initial laboratory-based studies of PPMOs in clinically-relevant infectious diseases hold promise for treating drug-resistant TB. Newer treatment options may also include interference with mycobacterial micro-RNAs [127].

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

A number of cases of TDR-TB have been reported; the causes are most probably multifactorial including host-associated variables, pharmacokinetics, co-infections and comorbidities. Several factors associated with the M.tb strain, including genetic and phenotypic changes, also contribute to the phenomenon of TDR-TB. Novel drugs are being developed, but will not eradicate resistance in M.tb. Thus, adjunct treatment options should be considered for patients with drug-resistant TB.

The clinical viewpoint from one of the most experienced TB hospitals in South Africa, reflecting the gap between clinical reality and academic ‘textbook’ views, is summarized in Box 1. The clinical experience in managing patients with drug-resistant TB highlights a multifactorial approach taking into consideration the cultural factors. As shown in Box 1, there are many challenges that need to be addressed to pro-actively deal with the management of drug-resistant TB.

Box 1. Clinical experience from King Dinuzulu Hospital (in Durban, province of KwaZulu–Natal), which is the leading South African provincial hospital for the treatment of drug-resistant TB

Clinicians’ viewpoint from the frontline of MDR-/XDR-TB patient management: the real-life clinical situation

Clinicians experience many challenges in the management of patients with drug-resistant TB. The success of treatment is often dependent on the following:

  • Quality of diagnosis and testing
  • Medication, side effects and drug regimens
  • The patient and the programme

Quality of diagnosis and testing

Through the decades of working in an MDR-/XDR-TB facility, clinicians face many confounding and conflicting results in day-to-day clinical practice. At times, they are confronted with radically different laboratory findings on drug sensitivity patterns obtained from sputum samples taken on consecutive or even the same day, assessed by standard culture techniques. Often these will have been performed at the same laboratory or at different laboratories having stringent quality controls. Recently, discordance between phenotypic and genotypic testing has been observed. A switch from a fully sensitive result, to isoniazid mono-resistance, to isoniazid and rifampicin resistance on genotypic testing, then reported as fully sensitive or total XDR patterns on full phenotypic sensitivity testing have also been observed on consecutive sputum samples from the same patient. Sometimes, these conflicting results have been encountered in totally asymptomatic patients. There have also been patients who have had intermittent findings of nontuberculosis mycobacterium interspersed with results of MDR-/XDR-TB. Some patients fail treatment, despite minimal resistance and good compliance. It is challenging to manage drug-resistant TB patients facing these conflicting results.

Many experts point out that this discordance is only observed in a small percentage of patients, but there are greater numbers of these patients and more apparent dilemmas in a country with a high burden of disease. Any nonoptimal treatment may have a negative impact on the individual patient in terms of increased morbidity or mortality. Several plausible reasons have been proposed for the discordant results, such as multiple anatomical sources causing multiple strains of M.tb within the same patient, selective mutations due to different degrees of bioavailability of anti-TB drugs in the different lesion sites, re-infection due to poor infection control in congregate settings, laboratory errors, poor quality of drugs, mixing of specimens at collection points or even fraudulent samples being presented to obtain a disability grant.

The consequence is uncertainty, and therefore the basis of treatment decisions is challenged. Without a doubt the new genotypic tests such as GeneXpert have detected far more patients. In theory, genetic testing should save more lives and reduce the costs of unnecessary treatment and consequent morbidity in some patients. The term ‘acceptable collateral damage’ has been applied to this situation.

Medication, side effects and drug regimens

Excellent treatment and drug regimens are the keys to achieve success in any MDR programme. The ideal treatment comprises a cheap, safe, high-quality medication, for a short duration, and with high efficacy. However, in reality, expensive medication is required for long periods with a large number of side effects. The consequence is often reluctance amongst patients to take toxic medications for the required duration. The essential component of the current intensive phase of chemotherapeutic regimen is long-term injectables. Many patients, who were productive members of society, have been reduced to deaf, unemployed patients, who become a burden to their families and the state as a consequence of treatment. Other patients, especially in countries with a high HIV burden, have a particular high likelihood of electrolyte imbalances and renal issues, requiring either hospitalization or intensive monitoring (which is difficult in a resource-poor setting). Other patients also suffer from neuropsychiatric side effects such as epilepsy, depression, suicidal ideation and peripheral neuropathy. These side effects are also more frequent in HIV-positive patients. Many of the newer drugs require more intensive electrocardiographic, and particularly QT interval, monitoring that has not been implemented routinely in many programmes. At present, effective treatment is often associated with many side effects and a long treatment duration. To change this scenario and to improve outcomes, novel, cheaper, effective drugs and drug combinations that need to be taken for short durations and with minimal side effects are urgently required.

The patient and the programme

Most effective TB programmes require patients to understand their disease and treatment plans, along with support to assist them to effectively complete their regimens. In many resource-poor high-burden settings, there is a lack of adequate counselling for patients and their families. There are often insufficient numbers of beds for sick patients who require hospital care. The impact of GeneXpert in certain areas has resulted in a dramatic increase in patient numbers, which has not been matched by an increase in the capacity for management by health care sectors. This has resulted in waiting lists for admission or for the initiation of M/XDR-TB treatment in some regions. In many areas, DOT programmes have failed and patients therefore take ‘self-supervised medication’. When patients fail treatment, there is never any certainty whether or not they have actually been adherent to treatment. Some regular attendees of MDR-TB units have been observed discarding their medication on their way out of the facility. The management of patients with MDR-TB requires some capacity at the district and regional level for supervision of treatment in a way that is acceptable to the patient and in a culturally appropriate context. Proper adherence depends on proper support of patients, monitoring of key outcomes and effective management of adverse effects.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References

This work was funded by HLF, Vinnova, VR, SIDA, Sweden and EDCTP (TBNeat) (to MM), and the UK Medical Research Council, European Union Framework7 Rid-RTI, European Developing Countries Clinical Trials Partnership (EDCTP), UBS Optimus Foundation, Switzerland and the NIHR Biomedical Research Centre, University College Hospitals, London, UK (to AZ).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Totally drug-resistant tuberculosis
  5. Clinical Mycobacterium tuberculosis spread and diagnosis
  6. Causes of TB resistance
  7. Mycobacterium tuberculosis genotype and drug resistance
  8. Whole-genome sequencing to define the genetic determinants of drug resistance
  9. Clinical in vitro nonresponsiveness
  10. Host versus drug-resistant Mycobacterium tuberculosis
  11. TDR-TB, co-infection and comorbidity
  12. Novel anti-TB drugs
  13. Adjunct treatments
  14. Repurposing of drugs
  15. Conclusion
  16. Conflict of interest statement
  17. Funding
  18. References
  • 1
    Pyle MM. Relative numbers of resistant tubercle bacilli in sputa of patients before and during treatment with streptomycin. Proc Staff Meet Mayo Clin 1947; 22: 46573.
  • 2
    Crofton J, Mitchison DA. Streptomycin resistance in pulmonary tuberculosis. Br Med J 1948; 2: 100915.
  • 3
    Medical Research Council (MRC). TREATMENT of pulmonary tuberculosis with streptomycin and para-aminosalicylic acid; a Medical Research Council investigation. Br Med J 1950; 2: 107385.
  • 4
    Fox W, Wiener A, Mitchison DA, Selkon JB, Sutherland I. The prevalence of drug-resistant tubercle bacilli in untreated patients with pulmonary tuberculosis; a national survey, 1955–56. Tubercle 1957; 38: 7184.
  • 5
    Mitchison DA, Selkon JB. Bacteriological aspects of a survey of the incidence of drug-resistant tubercle bacilli among untreated patients. Tubercle 1957; 38: 8598.
  • 6
    Crofton J. Chemotherapy of pulmonary tuberculosis. Br Med J 1959; 1: 16104.
  • 7
    Frieden TR, Sterling T, Pablos-Mendez A, Kilburn JO, Cauthen GM, Dooley SW. The emergence of drug-resistant tuberculosis in New York City. N Engl J Med 1993; 328: 5216.
  • 8
    Wright ABG, Barrera L, Boulahbal F et al. Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs – Worldwide, 2000–2004. MMWR Morb Mortal Wkly Rep 2006; 55: 3015.
  • 9
    Gandhi NR, Moll A, Sturm AW et al. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 2006; 368: 157580.
  • 10
    Migliori GB, Besozzi G, Girardi E et al. Clinical and operational value of the extensively drug-resistant tuberculosis definition. Eur Respir J 2007; 30: 6236.
  • 11
    World health organization (WHO). Global tuberculosis report 2013. WHO Library Cataloguing-in-Publication Data 2013 Global tuberculosis control Geneva, Switzerland: WHO/HTM/TB/2013.11
  • 12
    Dalton T, Cegielski P, Akksilp S et al. Prevalence of and risk factors for resistance to second-line drugs in people with multidrug-resistant tuberculosis in eight countries: a prospective cohort study. Lancet 2012; 380: 140617.
  • 13
    Migliori GB, De Iaco G, Besozzi G, Centis R, Cirillo DM. First tuberculosis cases in Italy resistant to all tested drugs. Euro Surveill 2007; 12: E070517.1.
  • 14
    Velayati AA, Masjedi MR, Farnia P et al. Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in Iran. Chest 2009; 136: 4205.
  • 15
    Udwadia ZF, Amale RA, Ajbani KK, Rodrigues C. Totally drug-resistant tuberculosis in India. Clin Infect Dis 2012; 54: 57981.
  • 16
    Udwadia Z, Vendoti D. Totally drug-resistant tuberculosis (TDR-TB) in India: every dark cloud has a silver lining. J Epidemiol Community Health 2013; 67: 4712.
  • 17
    Velayati AA, Farnia P, Ibrahim TA et al. Differences in cell wall thickness between resistant and nonresistant strains of Mycobacterium tuberculosis: using transmission electron microscopy. Chemotherapy 2009; 55: 3037.
  • 18
    Velayati AA, Farnia P, Merza MA et al. New insight into extremely drug-resistant tuberculosis: using atomic force microscopy. Eur Respir J 2010; 36: 14903.
  • 19
    Farnia P, Mohammad RM, Merza MA et al. Growth and cell-division in extensive (XDR) and extremely drug resistant (XXDR) tuberculosis strains: transmission and atomic force observation. Int J Clin Exp Med 2010; 3: 30814.
  • 20
    Klopper M, Warren RM, Hayes C et al. Emergence and spread of extensively and totally drug-resistant tuberculosis, South Africa. Emerg Infect Dis 2013; 19: 44955.
  • 21
    WHO. Multidrug and extensively drug-resistant TB (M/XDR-TB). 2010 Global report on surveillance and response. Geneva: World Health Organization, 2010.
  • 22
    Srivastava S, Sherman C, Gumbo T. In vitro susceptibility testing and totally drug-resistant tuberculosis. Eur Respir J 2013; 42: 2912.
  • 23
    Lawn SD, Mwaba P, Bates M et al. Advances in tuberculosis diagnostics: the Xpert MTB/RIF assay and future prospects for a point-of-care test. Lancet Infect Dis 2013; 13: 34961.
  • 24
    Jacobson KR, Theron D, Kendall EA et al. Implementation of genotype MTBDRplus reduces time to multidrug-resistant tuberculosis therapy initiation in South Africa. Clin Infect Dis 2013; 56: 5038.
  • 25
    Barnard M, Warren R, Gey Van Pittius N et al. Genotype MTBDRsl line probe assay shortens time to diagnosis of extensively drug-resistant tuberculosis in a high-throughput diagnostic laboratory. Am J Respir Crit Care Med 2012; 186: 1298305.
  • 26
    Baylan O, Kisa O, Albay A, Doganci L. Evaluation of a new automated, rapid, colorimetric culture system using solid medium for laboratory diagnosis of tuberculosis and determination of anti-tuberculosis drug susceptibility. Int J Tuberc Lung Dis 2004; 8: 7727.
  • 27
    Moore DA, Evans CA, Gilman RH et al. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med 2006; 355: 153950.
  • 28
    Pai M, Kalantri S, Pascopella L, Riley LW, Reingold AL. Bacteriophage-based assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis. J Infect 2005; 51: 17587.
  • 29
    Migliori GB, Matteelli A, Cirillo D, Pai M. Diagnosis of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis: current standards and challenges. Can J Infect Dis Med Microbiol 2008; 19: 16972.
  • 30
    Nosova E, Krasnova MA, Galkina K, Makarova MV, Litvinov VI, Moroz AM. Comparing performance of “TB-BIOCHIP”, “Xpert MTB/RIF” and “genotype MTBDRplus” assays for fast identification of mutations in the Mycobacterium tuberculosis complex in sputum from TB patients. Mol Biol (Mosk) 2013; 47: 26774.
  • 31
    McGrath M, Gey van Pittius NC, van Helden PD, Warren RM, Warner DF. Mutation rate and the emergence of drug resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 2014; 69: 292302.
  • 32
    Pasipanodya JG, Srivastava S, Gumbo T. Meta-analysis of clinical studies supports the pharmacokinetic variability hypothesis for acquired drug resistance and failure of antituberculosis therapy. Clin Infect Dis 2012; 55: 16977.
  • 33
    Grossman RF, Hsueh PR, Gillespie SH, Blasi F. Community-acquired pneumonia and tuberculosis: differential diagnosis and the use of fluoroquinolones. Int J Infect Dis 2014; 18: 1421.
  • 34
    Smith T, Wolff KA, Nguyen L. Molecular biology of drug resistance in Mycobacterium tuberculosis. Curr Top Microbiol Immunol 2013; 374: 5380.
  • 35
    Colijn C, Cohen T, Ganesh A, Murray M. Spontaneous emergence of multiple drug resistance in tuberculosis before and during therapy. PLoS One 2011; 6: e18327.
  • 36
    Comas I, Gagneux S. A role for systems epidemiology in tuberculosis research. Trends Microbiol 2011; 19: 492500.
  • 37
    Jenkins C, Bacon J, Allnutt J et al. Enhanced heterogeneity of rpoB in Mycobacterium tuberculosis found at low pH. J Antimicrob Chemother 2009; 63: 111820.
  • 38
    Walter ND, Strong M, Belknap R, Ordway DJ, Daley CL, Chan ED. Translating basic science insight into public health action for multidrug- and extensively drug-resistant tuberculosis. Respirology 2012; 17: 77291.
  • 39
    David HL. Probability distribution of drug-resistant mutants in unselected populations of Mycobacterium tuberculosis. Appl Microbiol 1970; 20: 8104.
  • 40
    Sandgren A, Strong M, Muthukrishnan P, Weiner BK, Church GM, Murray MB. Tuberculosis drug resistance mutation database. PLoS medicine 2009; 6: e2.
  • 41
    Kato-Maeda M, Ho C, Passarelli B et al. Use of whole genome sequencing to determine the microevolution of Mycobacterium tuberculosis during an outbreak. PLoS One 2013; 8: e58235.
  • 42
    Merker M, Kohl TA, Roetzer A et al. Whole genome sequencing reveals complex evolution patterns of multidrug-resistant Mycobacterium tuberculosis Beijing strains in patients. PLoS One 2013; 8: e82551.
  • 43
    Roetzer A, Diel R, Kohl TA et al. Whole genome sequencing versus traditional genotyping for investigation of a Mycobacterium tuberculosis outbreak: a longitudinal molecular epidemiological study. PLoS Med 2013; 10: e1001387.
  • 44
    Zhang H, Li D, Zhao L et al. Genome sequencing of 161 Mycobacterium tuberculosis isolates from China identifies genes and intergenic regions associated with drug resistance. Nat Genet 2013; 45: 125560.
  • 45
    Farhat MR, Shapiro BJ, Kieser KJ et al. Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis. Nat Genet 2013; 45: 11839.
  • 46
    Pepper DJ, Rebe K, Morroni C, Wilkinson RJ, Meintjes G. Clinical deterioration during antitubercular treatment at a district hospital in South Africa: the importance of drug resistance and AIDS defining illnesses. PLoS One 2009; 4: e4520.
  • 47
    Bottger EC. The ins and outs of Mycobacterium tuberculosis drug susceptibility testing. Clin Microbiol Infect 2011; 17: 112834.
  • 48
    Gagneux S, DeRiemer K, Van T et al. Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2006; 103: 286973.
  • 49
    Portevin D, Gagneux S, Comas I, Young D. Human macrophage responses to clinical isolates from the Mycobacterium tuberculosis complex discriminate between ancient and modern lineages. PLoS Pathog 2011; 7: e1001307.
  • 50
    European Concerted Action on New Generation Genetic Markers and Techniques for the Epidemiology and Control of Tuberculosis Beijing/W genotype Mycobacterium tuberculosis and drug resistance. Emerg Infect Dis 2006; 12: 73643.
  • 51
    Fleischmann RD, Alland D, Eisen JA et al. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol 2002; 184: 547990.
  • 52
    Lopez B, Aguilar D, Orozco H et al. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 2003; 133: 307.
  • 53
    Comas I, Chakravartti J, Small PM et al. Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved. Nat Genet 2010; 42: 498503.
  • 54
    Ford CB, Lin PL, Chase MR et al. Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection. Nat Genet 2011; 43: 4826.
  • 55
    Ford CB, Shah RR, Maeda MK et al. Mycobacterium tuberculosis mutation rate estimates from different lineages predict substantial differences in the emergence of drug-resistant tuberculosis. Nat Genet 2013; 45: 78490.
  • 56
    Marais BJ, Victor TC, Hesseling AC et al. Beijing and Haarlem genotypes are overrepresented among children with drug-resistant tuberculosis in the Western Cape Province of South Africa. J Clin Microbiol 2006; 44: 353943.
  • 57
    Toungoussova OS, Caugant DA, Sandven P, Mariandyshev AO, Bjune G. Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype. FEMS Immunol Med Microbiol 2004; 42: 28190.
  • 58
    Basile JI, Geffner LJ, Romero MM et al. Outbreaks of mycobacterium tuberculosis MDR strains induce high IL-17 T-cell response in patients with MDR tuberculosis that is closely associated with high antigen load. J Infect Dis 2011; 204: 105464.
  • 59
    Selwyn PA, Hartel D, Lewis VA et al. A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N Engl J Med 1989; 320: 54550.
  • 60
    Stevenson CR, Forouhi NG, Roglic G et al. Diabetes and tuberculosis: the impact of the diabetes epidemic on tuberculosis incidence. BMC Public Health 2007; 7: 234.
  • 61
    Jeon CY, Murray MB. Diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies. PLoS Med 2008; 5: e152.
  • 62
    Wu VC, Wang CY, Shiao CC et al. Increased risk of active tuberculosis following acute kidney injury: a nationwide, population-based study. PLoS One 2013; 8: e69556.
  • 63
    Skrahina A, Hurevich H, Zalutskaya A et al. Alarming levels of drug-resistant tuberculosis in Belarus: results of a survey in Minsk. Eur Respir J 2012; 39: 142531.
  • 64
    Dixon WG, Watson K, Lunt M, Hyrich KL, Silman AJ, Symmons DP. Rates of serious infection, including site-specific and bacterial intracellular infection, in rheumatoid arthritis patients receiving anti-tumor necrosis factor therapy: results from the British Society for Rheumatology Biologics Register. Arthritis Rheum 2006; 54: 236876.
  • 65
    Vento S, Lanzafame M. Tuberculosis and cancer: a complex and dangerous liaison. Lancet Oncol 2011; 12: 5202.
  • 66
    Stoffels K, Allix-Beguec C, Groenen G et al. From multidrug- to extensively drug-resistant tuberculosis: upward trends as seen from a 15-year nationwide study. PLoS One 2013; 8: e63128.
  • 67
    Maurya V, Vijayan VK, Shah A. Smoking and tuberculosis: an association overlooked. Int J Tuberc Lung Dis 2002; 6: 94251.
  • 68
    Bashar M, Alcabes P, Rom WN, Condos R. Increased incidence of multidrug-resistant tuberculosis in diabetic patients on the Bellevue Chest Service, 1987 to 1997. Chest 2001; 120: 15149.
  • 69
    Cox HS, Kalon S, Allamuratova S et al. Multidrug-resistant tuberculosis treatment outcomes in Karakalpakstan, Uzbekistan: treatment complexity and XDR-TB among treatment failures. PLoS One 2007; 2: e1126.
  • 70
    Johnston JC, Shahidi NC, Sadatsafavi M, Fitzgerald JM. Treatment outcomes of multidrug-resistant tuberculosis: a systematic review and meta-analysis. PLoS One 2009; 4: e6914.
  • 71
    Espinal MA, Laserson K, Camacho M et al. Determinants of drug-resistant tuberculosis: analysis of 11 countries. Int J Tuberc Lung Dis 2001; 5: 88793.
  • 72
    Singh S, Sankar MM, Gopinath K. High rate of extensively drug-resistant tuberculosis in Indian AIDS patients. AIDS 2007; 21: 23457.
  • 73
    Gurumurthy P, Ramachandran G, Hemanth Kumar AK et al. Decreased bioavailability of rifampin and other antituberculosis drugs in patients with advanced human immunodeficiency virus disease. Antimicrob Agents Chemother 2004; 48: 44735.
  • 74
    Mukherjee T, Boshoff H. Nitroimidazoles for the treatment of TB: past, present and future. Future Med Chem 2011; 3: 142754.
  • 75
    Manjunatha U, Boshoff HI, Barry CE. The mechanism of action of PA-824: novel insights from transcriptional profiling. Commun Integr Biol 2009; 2: 2158.
  • 76
    Matsumoto M, Hashizume H, Tomishige T et al. OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice. PLoS Med 2006; 3: e466.
  • 77
    Gler MT, Skripconoka V, Sanchez-Garavito E et al. Delamanid for multidrug-resistant pulmonary tuberculosis. N Engl J Med 2012; 366: 215160.
  • 78
    Andries K, Verhasselt P, Guillemont J et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005; 307: 2237.
  • 79
    Koul A, Vranckx L, Dendouga N et al. Diarylquinolines are bactericidal for dormant mycobacteria as a result of disturbed ATP homeostasis. J Biol Chem 2008; 283: 2527380.
  • 80
    Diacon AH, Pym A, Grobusch M et al. The diarylquinoline TMC207 for multidrug-resistant tuberculosis. N Engl J Med 2009; 360: 2397405.
  • 81
    Voelker R. MDR-TB has new drug foe after fast-track approval. JAMA 2013; 309: 430.
  • 82
    Protopopova M, Hanrahan C, Nikonenko B et al. Identification of a new antitubercular drug candidate, SQ109, from a combinatorial library of 1,2-ethylenediamines. J Antimicrob Chemother 2005; 56: 96874.
  • 83
    Tahlan K, Wilson R, Kastrinsky DB et al. SQ109 targets MmpL3, a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2012; 56: 1797809.
  • 84
    Migliori GB, Sotgiu G, Gandhi NR et al. Drug resistance beyond extensively drug-resistant tuberculosis: individual patient data meta-analysis. Eur Respir J 2013; 42: 16979.
  • 85
    Reddy VM, Einck L, Andries K, Nacy CA. In vitro interactions between new antitubercular drug candidates SQ109 and TMC207. Antimicrob Agents Chemother 2010; 54: 28406.
  • 86
    Chen P, Gearhart J, Protopopova M, Einck L, Nacy CA. Synergistic interactions of SQ109, a new ethylene diamine, with front-line antitubercular drugs in vitro. J Antimicrob Chemother 2006; 58: 3327.
  • 87
    Swaney SM, Aoki H, Ganoza MC, Shinabarger DL. The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob Agents Chemother 1998; 42: 32515.
  • 88
    Lee M, Lee J, Carroll MW et al. Linezolid for treatment of chronic extensively drug-resistant tuberculosis. N Engl J Med 2012; 367: 150818.
  • 89
    Sotgiu G, Centis R, D'Ambrosio L, Spanevello A, Migliori GB. Linezolid to treat extensively drug-resistant TB: retrospective data are confirmed by experimental evidence. Eur Respir J 2013; 42: 28890.
  • 90
    Shaw KJ, Barbachyn MR. The oxazolidinones: past, present, and future. Ann N Y Acad Sci 2011; 1241: 4870.
  • 91
    Alffenaar JW, van der Laan T, Simons S et al. Susceptibility of clinical Mycobacterium tuberculosis isolates to a potentially less toxic derivate of linezolid, PNU-100480. Antimicrob Agents Chemother 2011; 55: 12879.
  • 92
    Balasubramanian V, Solapure S, Iyer H et al. Bactericidal activity and mechanism of action of AZD5847: a novel oxazolidinone for the treatment of tuberculosis. Antimicrob Agents Chemother 2014; 58: 495502.
  • 93
    Zumla A, Nahid P, Cole ST. Advances in the development of new tuberculosis drugs and treatment regimens. Nat Rev Drug Discovery 2013; 12: 388404.
  • 94
    Reddy VM, Dubuisson T, Einck L et al. SQ109 and PNU-100480 interact to kill Mycobacterium tuberculosis in vitro. J Antimicrob Chemother 2012; 67: 11636.
  • 95
    Wallis RS, Jakubiec W, Mitton-Fry M et al. Rapid evaluation in whole blood culture of regimens for XDR-TB containing PNU-100480 (sutezolid), TMC207, PA-824, SQ109, and pyrazinamide. PLoS One 2012; 7: e30479.
  • 96
    Pethe K, Bifani P, Jang J et al. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat Med 2013; 19: 115760.
  • 97
    Vilaplana C, Montane E, Pinto S et al. Double-blind, randomized, placebo-controlled Phase I Clinical Trial of the therapeutical antituberculous vaccine RUTI. Vaccine 2010; 28: 110616.
  • 98
    Katoch K, Singh P, Adhikari T et al. Potential of Mw as a prophylactic vaccine against pulmonary tuberculosis. Vaccine 2008; 26: 122834.
  • 99
    Dlugovitzky D, Stanford C, Stanford J. Immunological basis for the introduction of immunotherapy with Mycobacterium vaccae into the routine treatment of TB. Immunotherapy 2011; 3: 55768.
  • 100
    Skinner MA, Yuan S, Prestidge R, Chuk D, Watson JD, Tan PL. Immunization with heat-killed Mycobacterium vaccae stimulates CD8+ cytotoxic T cells specific for macrophages infected with Mycobacterium tuberculosis. Infect Immun 1997; 65: 452530.
  • 101
    Prabowo SA, Groschel MI, Schmidt ED et al. Targeting multidrug-resistant tuberculosis (MDR-TB) by therapeutic vaccines. Med Microbiol Immunol 2013; 202: 95104.
  • 102
    Coler RN, Bertholet S, Pine SO et al. Therapeutic immunization against Mycobacterium tuberculosis is an effective adjunct to antibiotic treatment. J Infect Dis 2013; 207: 124252.
  • 103
    Aagaard C, Hoang T, Dietrich J et al. A multistage tuberculosis vaccine that confers efficient protection before and after exposure. Nat Med 2011; 17: 18994.
  • 104
    Grode L, Ganoza CA, Brohm C, Weiner J III, Eisele B, Kaufmann SH. Safety and immunogenicity of the recombinant BCG vaccine VPM1002 in a phase 1 open-label randomized clinical trial. Vaccine 2013; 31: 13408.
  • 105
    Malowany JI, McCormick S, Santosuosso M et al. Development of cell-based tuberculosis vaccines: genetically modified dendritic cell vaccine is a much more potent activator of CD4 and CD8 T cells than peptide- or protein-loaded counterparts. Mol Ther 2006; 13: 76675.
  • 106
    Singh V, Jain S, Gowthaman U et al. Co-administration of IL-1+IL-6+TNF-alpha with Mycobacterium tuberculosis infected macrophages vaccine induces better protective T cell memory than BCG. PLoS One 2011; 6: e16097.
  • 107
    Skrahin A, Ahmed RK, Ferrara G et al. Autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an open-label phase 1 safety trial. Lancet Respir Med 2014; 2: 10822.
  • 108
    Martineau AR, Wilkinson RJ, Wilkinson KA et al. A single dose of vitamin D enhances immunity to mycobacteria. Am J Respir Crit Care Med 2007; 176: 20813.
  • 109
    Coussens AK, Wilkinson RJ, Hanifa Y et al. Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment. Proc Natl Acad Sci USA 2012; 109: 1544954.
  • 110
    Martineau AR, Wilkinson KA, Newton SM et al. IFN-gamma- and TNF-independent vitamin D-inducible human suppression of mycobacteria: the role of cathelicidin LL-37. J Immunol 2007; 178: 71908.
  • 111
    Hossein-nezhad A, Holick MF. Optimize dietary intake of vitamin D: an epigenetic perspective. Curr Opin Clin Nutr Metab Care 2012; 15: 56779.
  • 112
    Baeke F, Takiishi T, Korf H, Gysemans C, Mathieu C. Vitamin D: modulator of the immune system. Curr Opin Pharmacol 2010; 10: 48296.
  • 113
    Liu PT, Schenk M, Walker VP et al. Convergence of IL-1beta and VDR activation pathways in human TLR2/1-induced antimicrobial responses. PLoS One 2009; 4: e5810.
  • 114
    Sonawane A, Santos JC, Mishra BB et al. Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages. Cell Microbiol 2011; 13: 160117.
  • 115
    Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004; 119: 75366.
  • 116
    Yuk JM, Shin DM, Lee HM et al. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Cell Host Microbe 2009; 6: 23143.
  • 117
    Wejse C, Gomes VF, Rabna P et al. Vitamin D as supplementary treatment for tuberculosis: a double-blind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 2009; 179: 84350.
  • 118
    Martineau AR, Timms PM, Bothamley GH et al. High-dose vitamin D(3) during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial. Lancet 2011; 377: 24250.
  • 119
    Vilcheze C, Hartman T, Weinrick B, Jacobs WR Jr. Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction. Nat Commun 2013; 4: 1881.
  • 120
    Guzman JD, Evangelopoulos D, Gupta A et al. Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay. BMJ Open 2013; 3: e002672.
  • 121
    Gupta S, Cohen KA, Winglee K, Maiga M, Diarra B, Bishai WR. Efflux inhibition with verapamil potentiates bedaquiline in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2014; 58: 5746.
  • 122
    Gupta S, Tyagi S, Almeida DV, Maiga MC, Ammerman NC, Bishai WR. Acceleration of tuberculosis treatment by adjunctive therapy with verapamil as an efflux inhibitor. Am J Respir Crit Care Med 2013; 188: 6007.
  • 123
    Tilley LD, Hine OS, Kellogg JA et al. Gene-specific effects of antisense phosphorodiamidate morpholino oligomer-peptide conjugates on Escherichia coli and Salmonella enterica serovar typhimurium in pure culture and in tissue culture. Antimicrob Agents Chemother 2006; 50: 278996.
  • 124
    Greenberg DE, Marshall-Batty KR, Brinster LR et al. Antisense phosphorodiamidate morpholino oligomers targeted to an essential gene inhibit Burkholderia cepacia complex. J Infect Dis 2010; 201: 182230.
  • 125
    Geller BL, Marshall-Batty K, Schnell FJ, McKnight MM, Iversen PL, Greenberg DE. Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo. J Infect Dis 2013; 208: 155360.
  • 126
    Lai BS, Witola WH, El Bissati K et al. Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections. Proc Natl Acad Sci USA 2012; 109: 141827.
  • 127
    Ma F, Xu S, Liu X et al. The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-gamma. Nat Immunol 2011; 12: 8619.
  • 128
    Koser CU, Bryant JM, Becq J et al. Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med 2013; 369: 2902.