I. Tuberculosis 1. Etiology and historical perspective

Tuberculosis is a disease caused by Mycobacterium tuberculosis, a bacterium belonging to the genus Mycobacterium, the family Mycobacteriaceae, the order Actinomycetales, and the class of Actinobacteria. M. tuberculosis belongs to the M. tuberculosis complex, which originally groups mycobacteria with similar biological characteristics and being source of human disease with similar clinical features. Two other mycobacteria belonging to the M. tuberculosis complex are responsible of clinically-relevant forms of diseases in humans: M. africanum and M. bovis. The other mycobacteria belonging to this group are mainly animal pathogens.

Tuberculosis is an ancestral disease that has been accompanying the history of humankind since its beginning. Initial theories identified the transition from nomadism to the first settlement of human tribes and the beginning of animal herding (approximately 8 000 years B. C.) as the moment of the first appearance of tuberculosis, thus supporting the hypothesis that M. tuberculosis would have originated from M. bovis transmitted from cattle to men. However, the study of whole genome sequence of the different mycobacteria has led to reject this hypothesis, rather suggesting that M. tuberculosis complex bacteria share a common ancestor that was similar to M. tuberculosis and could probably have been a human pathogen already (Figure 1). [START_REF] Brosch | A new evolutionary scenario for the Mycobacterium tuberculosis complex[END_REF] This is supported by paleomicrobiology studies that have identified ancient DNA resembling M. tuberculosis genome on mummies with tuberculosislike lesions. [START_REF] Donoghue | Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA[END_REF] The first known evidence of human tuberculosis comes from a Homo erectus skull with the signs of tuberculosis meningitis found near Ankara, Turkey, and dating approximately from 500 000 years B. C. [START_REF] Kappelman | First Homo erectus from Turkey and implications for migrations into temperate Eurasia[END_REF] It is therefore likely that the ancestor of M. tuberculosis was a human pathogen that was present well before human tribes settled down.

The first descriptions of the clinical features of tuberculosis disease, called phthisis, were provided by Greek physicians Hyppocrates and, later, Galenus. It was during this time that the contagious nature of tuberculosis was hypothetized for the first time. During Middle Age, this theory was abandoned and replaced by different theories and beliefs, including the hereditary nature of tuberculosis, or at least a genetic predisposition ("constitutional disease"), or the association of tuberculosis to the accumulation of chemical products in the human body ("zymotic fever"). In the XVI and XVII centuries, the establishment of human dissection led to the first descriptions of the anatomopathological characteristics of tuberculosis diseases, including the definition of "tubercle". In the XVIII and XIX centuries, the tuberculosis epidemic raged through Europe, taking advantage of the increasing urbanisation and thriving during the Industrial Revolution. It is only in 1882 that a German scientist, Robert Koch, produced the irrefutable proof that tuberculosis is a contagious disease by isolating M. tuberculosis bacteria. The search for preventive, diagnostic, and therapeutic tools against tuberculosis could finally begin.

Epidemiology

According to the World Health Organization (WHO), in 2016 there were an estimated 10.4 million (8.8 -12.2 million) new cases of tuberculosis, equivalent to a global incidence rate of 140 per 100 000 population. The WHO has identified 30 high tuberculosis burden countries that account together for 87% of all estimated cases. In 2016, three countries accounted for 45% of cases worldwide: India, Indonesia, and China. An estimated 10% (8 -12%) of the incident cases occurred among people living with HIV, mostly concentrated in the WHO African Region and in particular in southern Africa. Overall, the incidence rate is decreasing very slowly, at an estimated pace of 1.9% in year 2016. In 2016, tuberculosis caused an estimated 1.3 million (1.2 -1.4 million) deaths in HIV-negative and an additional 374 000 (325 000 -427 000) deaths in HIV-positive patients. The mortality burden, the highest globally for a single infectious agent, and the ninth leading cause of death overall, is particularly striking if we considered that this disease is curable and partially preventable.

Global treatment success rate among drug-susceptible cases rate was 83% in 2015. [START_REF] Who | Global tuberculosis report[END_REF] Overall, these impressive figures explain why tuberculosis is considered a global health priority.

Nevertheless, despite undeniable progress achieved in the last years, the efforts to control tuberculosis have been hindered by insufficient funding and scarce political commitment.

The WHO Europe Region accounts for 3% of global tuberculosis cases, with an incidence rate of 32 (27 -36) cases per 100 000 population. Overall, the incidence rate is decreasing faster that in other WHO Regions (4.6% since 2015). [START_REF] Who | Global tuberculosis report[END_REF] However, there is a strong gradient in tuberculosis burden between the European Union and the other Eastern countries that are part of the Europe Region, which account for the vast majority of cases and deaths.

In France, the total number of cases has been declining progressively since the first available data from the post-Second World War period, when the incidence was above 100 cases per 100 000 population, except for a transitory increase in the first years of 1990. France is currently considered a low-incidence country for tuberculosis, with 5 200 (4 500 -5 800) estimated cases and an incidence rate of 8 (7 -9) cases per 100 000 population in 2016 (Figure 2). [START_REF] Who | Global tuberculosis report[END_REF] The most recently available data on tuberculosis treatment outcomes in France are related to the 2009 cohort. Among pulmonary tuberculosis patients notified in 2009, the treatment success rate was 70%, significantly higher among smear-negative (73%) than among smear-positive (67%) cases. Overall, 30% of patients had unfavourable outcomes, including 10% deaths. 5 Source: Mitchison et al. [START_REF] Mitchison | The Diagnosis and Therapy of Tuberculosis During the Past 100 Years[END_REF]

Principles of tuberculosis treatment

The birth of antibacterial chemotherapy dates back to the discovery of penicillin in 1929 and its implementation in the 40s. Penicillin, however, had no activity on M.

tuberculosis and this underlined the first hurdle in anti-tuberculosis chemotherapy: the high level of natural resistance. This characteristic, common to all mycobacteria, is due to multiple mechanisms, including the production of inactivation enzymes like beta-lactamases, [START_REF] Jarlier | Interplay of Cell Wall Barrier and r-Lactamase Activity Determines High Resistance to 3-Lactam Antibiotics in Mycobacterium chelonae[END_REF] the expressions of efflux pumps who specifically target antibiotics, [START_REF] Sander | Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance[END_REF] and the natural low permeability of the mycobacterial cell wall. [START_REF] Jarlier | Mycobacterial cell wall: structure and role in natural resistance to antibiotics[END_REF] The first antimycobacterial, streptomycin, was discovered in 1943: the results of the first clinical trials were spectacular in both the rapid clinical response and the early appearance of resistance, leading to treatment failure or relapse in the vast majority of cases. [START_REF] Crofton | Streptomycin resistance in pulmonary tuberculosis[END_REF] Similar results were obtained with monotherapy with PAS, the second anti-tuberculosis compound, which was developed in 1944. [START_REF] Lehmann | Para-Aminosalicylic Acid in the treatment of Tuberculosis[END_REF] Conversely, the association of these two drugs was much more effective in preventing drug resistance. [START_REF] Tempel | Combined intermittent regimens employing streptomycin and para-aminosalicylic acid in the treatment of pulmonary tuberculosis; a comparison with daily and intermittent dosage schedules[END_REF] These observations have been the basis for the formulation of the first principle of anti-tuberculosis chemotherapy: never treat tuberculosis with a single drug. Isoniazid, the third antituberculosis drug and by far the most active at the time, was discovered a few years later, in 1951. [START_REF] Fox | Studies on the treatment of tuberculosis undertaken by the British Medical Research Council Tuberculosis Units[END_REF] This triple therapy would become the standard of care for tuberculosis treatment for almost 20 years. Multiple clinical trials established that the minimal duration of this regimen, in order to prevent relapses, was of 18 months. [START_REF] Murray | Treatment of Tuberculosis. A Historical Perspective[END_REF] This represents the second principle of antituberculosis chemotherapy: the treatment duration, typically much longer than that of any other bacterial infection, is linked to the intrinsic characteristics of the drugs included in the regimen. Indeed, when rifampicin was discovered in 1957 and implemented in the treatment in 1966, it allowed the total duration of treatment to be decreased to nine months. [START_REF] Maggi | Rifampicin: a new orally active rifamycin[END_REF] Finally, the addition of pyrazinamide, a drug that was discovered in the 40s but initially discarded due to high rates of adverse events, allowed for reduction of treatment to six months. This finding, demonstrated by a clinical trial by the British Medical Research Council in Singapore, posed the foundations of the so-called shortcourse regimen that is still the standard-of-care today, with the only change of ethambutol replacing streptomycin for safety and feasibility reasons. [START_REF] Tuberculosis | Clinical trial of six-month and four-month regimens of chemotherapy in the treatment of pulmonary tuberculosis[END_REF] The crucial role of rifampicin and pyrazinamide in shortening treatment duration was defined as sterilising activity. This latter, in contrast to bactericidal activity, is difficult to assess. Indeed, the difficulty in eradicating M. tuberculosis infection appears to be linked to the presence of different mycobacterial populations in the affected tissue (Figure 3). In particular, mycobacterial populations may have different degree of metabolic activity, from active replication to dormancy. Dormant mycobacteria are less susceptible to antimicrobials and likely susceptible only to sterilizing drugs like rifampicin and pyrazinamide. [START_REF] Wayne | An In Vitro Model for Sequential Study of Shiftdown of Mycobacterium tuberculosis through Two Stages of Nonreplicating Persistence[END_REF] Furthermore, specific populations of M. tuberculosis cells that do not grow in culture unless stimulated have been isolated in sputum of patients before starting anti-tuberculosis treatment.

These mycobacteria appear to have a high tolerance to all drugs, including sterilizing ones, and to acquire a spore-like structure. [START_REF] Shleeva | Dormant ovoid cells of Mycobacterium tuberculosis are formed in response to gradual external acidification[END_REF] Further studies have allowed to elucidate the sterilization activity of the rifampicin and pyrazinamide: while the former seems linked to the speed with which rifampicin kills mycobacteria as soon as they recover from dormancy, rather than in a particularly rapid killing of slowly-growing populations, [START_REF] Dickinson | Experimental models to explain the high sterilizing activity of rifampin in the chemotherapy of tuberculosis[END_REF] the latter appears to be consequent to a direct action on non-replicating mycobacteria in acidic environment. This hypothesis is confirmed by the fact that pyrazinamide sterilizing activity appears to be only exerted during the first months of treatment, waning in correspondence with the reduction of inflammation and the consequent increase of pH in the lesions. [START_REF] Hu | Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis[END_REF] The two-phase structure of anti-tuberculosis treatment, theorized for the first time in 1961 by Cannetti, [START_REF] Canetti | L'éradication de la tuberculose dnas les différents pays, compte tenu des conditions existantes (problèmes théoriques et solutions pratiques)[END_REF] was also established following the Singapore trial, comprising a first fourdrug intensive phase and the following continuation phase with the two most effective drugs. [START_REF] Mitchison | The chemotherapy of tuberculosis: past, present and future [State of the art[END_REF]

Surgery in tuberculosis treatment

Surgical treatment of tuberculosis was born long before the first anti-tuberculosis drug was discovered. The first surgical technique which was applied to patients with pulmonary forms of tuberculosis was the therapeutic pneumothorax, developed by Forlanini in 1882. This technique consisted in collapsing the lung affected by tuberculosis through injection of gas between visceral and parietal pleura. This intervention, although burdened by frequent and severe adverse events, allowed to cure multiple tuberculosis cases before the birth of antimycobacterial chemotherapy. [START_REF] Garbarino | A century ago: Carlo Forlanini and the first successful treatment of tuberculosis[END_REF] Between the end of the 19 th and the beginning of the 20 th century, thoracoplasty was introduced to treat tuberculosis patients with lung cavities. This technique is still used in cases where pulmonary resection is not feasible (i.e. patients with severe respiratory insufficiency or bilateral pulmonary lesions) or to treat pleural complications of previous pulmonary resections. [START_REF] Bai | Surgical treatment efficacy in 172 cases of tuberculosis-destroyed lungs[END_REF] The technique of pulmonary resection evolved in correspondence with the development of the first anti-tuberculosis agents. In fact, until 1970, lung surgery was performed in almost every case that was considered operable, together with medical treatment. [START_REF] Shields | Changing role of surgery in the treatment of pulmonary tuberculosis[END_REF] Subsequently, with the availability of rifampicin and based on historical data showing good outcome of patients receiving medical-only treatment, surgery ceased to be routinely performed in drug-susceptible tuberculosis. [START_REF] Neptune | Current surgical management of pulmonary tuberculosis[END_REF] With the rise of drug-resistant tuberculosis, the use of surgery has progressively regained momentum. In light of the abysmal outcomes achieved in the treatment of M/XDR-TB with medical treatment, surgery has been regarded as a promising adjuvant approach. In particular, surgery could have an important role on large cavitary lesions or on lung tissue with diffuse alterations of the parenchyma, where drug penetration is likely to be insufficient.

Currently, there is consensus on the following indications for surgery:

• Localised pulmonary lesions in a patient with positive sputum smear or culture exams after a few months of well-conducted treatment;

• Localised pulmonary lesions and high risk of treatment failure based on radiological presentation and mycobacterial resistance profile. [START_REF] Kemper | Surgical treatment of drug-resistant tuberculosis[END_REF] It is also recommended that the indication for surgery should be discussed by a multidisciplinary team including clinicians, surgeons, and other specialists. The timing of surgery is not completely established. It is however desirable, if possible, to wait for sputum culture conversion, or at least for a few months of effective treatment, to limit the risk of intra-surgical bacterial dissemination.

Two systematic reviews have been published that evaluate use of surgery in the treatment of M/XDR-TB, both considering studies from the late 70s up to 2012. In the first meta-analysis, 35 studies were retrieved, describing 1686 patients: overall, the rate of postsurgery complications was comprised between 0% and 29%, the mortality rate was between 0% and 8%, and the rate of successful outcome between 47% and 100%. [START_REF] Mordant | Chirurgie et tuberculose multi/ultrarésistante : une revue de la littérature réhabilite une intervention adjuvante à l'antibiothérapie chez des patients sélectionnés[END_REF] In the second systematic review, 47 publications were included. In 24 of them, the outcome of patients receiving medical treatment only or medical treatment plus surgery were compared: the metaanalysis found a significant association between surgery and treatment success. [START_REF] Marrone | Surgical interventions for drug-resistant tuberculosis: a systematic review and meta-analysis [Review article[END_REF] Of note, most of these studies have major methodological flaws, including the retrospective, observational nature, and in particular a very strong selection bias towards patients chosen to perform surgery. In addition, the post-surgical follow-up of patients is often limited and complicates the assessment the functional consequences of pulmonary resections.

In addition, a large meta-analysis of individual patient data, including 4238 patients from 18 surgical studies and 2193 patients from 8 nonsurgical studies, has been recently published. This study has provided the first good-quality indications on the best time and type of surgical intervention that should be used in the treatment of M/XDR-TB. In this metaanalysis, partial lung resection, rather then pneumonectomy, was shown to be associated with improved treatment outcome; in addition, treatment success was more likely when surgery was performed after culture conversion than before conversion. [START_REF] Fox | Surgery as an Adjunctive Treatment for Multidrug-Resistant Tuberculosis: An Individual Patient Data Metaanalysis[END_REF] However, the exact timing and indication for surgery are still not well established and highly variable across different centres.

Rates of rifampicin-resistant/multidrug-resistant tuberculosis are expressed among new tuberculosis cases (above) and previously treated tuberculosis cases (below). Source: World Health Organization,

Global TB Report 2017. [START_REF] Who | Global tuberculosis report[END_REF]

II. Drug-resistant tuberculosis

Epidemiology of drug-resistant tuberculosis

Drug-resistant tuberculosis is a growing global concern. In 2016, there were an estimated 490,000 new MDR-TB patients and 600,000 new cases of rifampin-resistant tuberculosis, with 240,000 total deaths among these two groups (Figure 4). [START_REF] Who | Global tuberculosis report[END_REF] Among MDR-TB patients tested for any fluoroquinolone, 20% (95% CI: 14-26%) had fluoroquinolone resistance. Consistently, in a recent multicountry survey, resistance to ofloxacin was present in 12.3% to 30.7% of rifampin-resistant TB patients, according to different settings. [START_REF] Zignol | Population-based resistance of Mycobacterium tuberculosis isolates to pyrazinamide and fluoroquinolones: results from a multicountry surveillance project[END_REF] Globally, the average proportion of MDR-TB patients harbouring XDR-TB strains was 6.2% (95% CI: 3.6-9.5%). Of the 600,000 estimated, new rifampin-resistant cases in 2016, only 153,119 (26%) were actually diagnosed with the disease. Fewer, 129,689 (22%), ever received any treatment with second-line TB drugs. [START_REF] Who | Global tuberculosis report[END_REF] These numbers could be even worse for XDR-TB, as only 39% of all detected MDR-TB cases performed drug susceptibility testing for both fluoroquinolones and second-line injectables. In 2015, only 7579 XDR-TB cases were reported and 7234 were treated. [START_REF] Who | Global tuberculosis report[END_REF] The WHO European region is considered to be the epicentre of the MDR-TB epidemic. It is estimated that, globally, 4.1% (95% CI: 2.8-5.3%) of new cases and 19% (95% CI: 9.8-27%) of previously treated cases had rifampicin-resistant tuberculosis in 2016: in the WHO European region, these two figures reached 19% (95% CI: 12-26%) and 55% (43-67%), respectively.

In France, the surveillance of drug-resistant tuberculosis is performed by the National Reference Center of Mycobacteria (CNR-MyRMA) since 1992. [START_REF] Robert | La résistance aux antituberculeux en France à travers les données des réseaux Azay-Mycobactéries et du Centre National de Référence des Mycobactéries[END_REF] In particular, the CNR-MyRMA hosts the only laboratory performing phenotypic drug susceptibility testing for second-line anti-tuberculosis drugs at a national level and participates to international surveillance networks. The annual number of rifampicin-resistant and M/XDR-TB cases in France has historically been stable around approximately 50 cases per year. [START_REF] Robert | Multidrug-resistant tuberculosis: eight years of surveillance in France[END_REF][START_REF] Robert | Surveillance of Mycobacterium tuberculosis drug resistance in France, 1995-1997[END_REF][START_REF] Meyssonnier | Rifampicin monoresistant tuberculosis in France: a 2005-2010 retrospective cohort analysis[END_REF] In the last decade, a surge of drug-resistant cases has been reported in association with migrant fluxes from Eastern Europe, Central Asia, and the Caucasus countries. [START_REF] Bernard | A surge of MDR and XDR tuberculosis in France among patients born in the Former Soviet Union[END_REF] In 2017, 82 MDR-TB strains were received by the CNR-MyRMA, 10 of which were classified as XDR-TB cases. 38 Rate of resistant strains emerged while on monotherapy with streptomycin (above) and, in parallel, with streptomycin alone, PAS alone, and streptomycin plus PAS (below). Source: Crofton et al 10 and Tempel et al. 12

Development of drug resistance

The development of drug resistance is a feature of most infectious agents and mycobacteria make no exception. Indeed, drug resistance appeared quickly during the first clinical trials assessing tuberculosis treatment. In the British Medical Research Council trial testing monotherapy with streptomycin, half of the patients had M. tuberculosis strains with drug resistance less than two months after treatment start. [START_REF] Crofton | Streptomycin resistance in pulmonary tuberculosis[END_REF] In a second trial, the addition of a second anti-tuberculosis agent like PAS allowed to prevent the emergence of drug resistance (Figure 5). [START_REF] Tempel | Combined intermittent regimens employing streptomycin and para-aminosalicylic acid in the treatment of pulmonary tuberculosis; a comparison with daily and intermittent dosage schedules[END_REF] Mycobacteria have no mechanism to transmit drug resistance (i.e. plasmids), which conversely are common in other bacteria. Resistance in M. tuberculosis is conferred by chromosomal mutations. Chromosomal mutations appear spontaneously at a rate that is different for each drug, ranging for example between 10 -8 for rifampicin and 10 -5 for streptomycin: this rate represents the resistance threshold of the drug and is a key element in the selection of drug resistance. The second main factor driving the in vivo selection of resistance is the bacterial burden present in the body of individual patients: it is generally estimated that patients with moderate lung involvement with infiltrates may harbour around 10 6 mycobacteria, while lung cavities may contain from 10 9 up to 10 12 mycobacteria. In the classic model of drug resistance genesis, spontaneously-appeared resistant mutants are selected by the exposure of the bacterial population to the drug, which kills susceptible isolates, and select for the resistant ones; progressive exposure to different drugs could eventually lead to the development of multidrug-resistance (Figure 6). 39 Schematic representing drug resistance selection during isoniazid monotherapy, followed by treatment with isoniazid plus rifampicin. Source: Dheda et al. [START_REF] Dheda | Global control of tuberculosis: from extensively drug-resistant to untreatable tuberculosis[END_REF] While this model is universally recognised and is adequate to explain the development of drug resistance in cases where suboptimal treatments are used (i.e. patients exposed to a single drug), a much more debated topic is the cause of the emergence of drug-resistance in patients receiving an adequate combination therapy. A common explanation is the scarce treatment adherence by the patient. This theory has led the tuberculosis national programs to include different tools to monitor treatment compliance, like directly-observed therapy.

However, in vitro hollow-fibre model studies have repeatedly failed to show a strong association between either missing treatment doses or taking them at different times, rather than all together (the so-called PK mismatch), and the selection of drug resistance. [START_REF] Srivastava | Multidrug-Resistant Tuberculosis Not Due to Noncompliance but to Between-Patient Pharmacokinetic Variability[END_REF][START_REF] Srivastava | Pharmacokinetic Mismatch Does Not Lead to Emergence of Isoniazid-or Rifampin-Resistant Mycobacterium tuberculosis but to Better Antimicrobial Effect: a New Paradigm for Antituberculosis Drug Scheduling[END_REF] Conversely, the same model was able to predict that interindividual variability in pharmacokinetics of the administered drugs, due to alterations in absorption and metabolism, would lead to the development of drug resistance in 0.68% of patients even in case of perfect treatment adherence. This finding was confirmed in vivo in a prospective cohort study in South Africa, where among 142 tuberculosis patients, 0.7% developed drug resistance despite good adherence after two months of treatment. All cases of acquired drug resistance and more than 90% of the cases of treatment failure were associated with low blood levels of the drugs. [START_REF] Pasipanodya | Serum Drug Concentrations Predictive of Pulmonary Tuberculosis Outcomes[END_REF] Another element predisposing the acquisition of drug resistance is the penetration properties of drugs into lung cavities, which has been shown to be highly variable between different compounds. [START_REF] Prideaux | The association between sterilizing activity and drug distribution into tuberculosis lesions[END_REF] The relevance of the variability of drug concentrations is confirmed by the finding that M. tuberculosis strains in different areas of lung cavities harbour different resistance-conferring mutations and different MICs for drugs being administered. [START_REF] Dheda | Drug Penetration Gradients Associated with Acquired Drug Resistance in Tuberculosis Patients[END_REF] The lineage of M. tuberculosis strains also appears to play a role in the acquisition of drug resistance. For instance, it has been demonstrated that strains belonging to lineage two (East Asian, including the Beijing strain that is clonally expanding in association with the spread of MDR-TB) have a higher spontaneous mutation rate than strains belonging to lineage four (Euro-America), and are therefore more likely to acquire resistance. [START_REF] Ford | Mycobacterium tuberculosis mutation rate estimates from different lineages predict substantial differences in the emergence of drug-resistant tuberculosis[END_REF] Finally, the role of efflux pumps in the selection of drug resistance remains to be elucidated: it has been postulated that efflux pumps genes may be induced after a few hours of drug pressure, leading to a status of low-level resistance that in turn might favour the selection of the resistant mutants. [START_REF] Dheda | Global control of tuberculosis: from extensively drug-resistant to untreatable tuberculosis[END_REF] Resistance-conferring mutations were historically thought to come with a fitness cost, leading for example to a reduced multiplication rate and to the progressive disappearance of the resistant sub-population in absence of selective pressure by chemotherapy. This theory has received multiple experimental confirmations. [START_REF] Andersson | The biological cost of antibiotic resistance[END_REF] However, it has been recently shown that compensatory mutations may appear in drug-resistant strains, leading to a high competitive fitness both in vitro and in vivo. [START_REF] Comas | Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes[END_REF] Similarly, drug-resistant tuberculosis has been traditionally labelled as a man-made disease and the direct consequence of suboptimal regimens or insufficient treatment adherence. The emphasis was therefore placed on the prevention of cases of secondary resistance and the presence of risk factors, such as previous treatment with first-or secondline anti-tuberculosis drugs, was regarded as the main predictor of drug-resistant tuberculosis.

However, this paradigm has been challenged by the increasing circulation of M/XDR-TB strains in the population. A recent modelling study has estimated that primary drug resistance transmission would account for a median of 95.9% (95% uncertainty range 68.0 -99.6) of all incident MDR-TB cases and 61.3% (16.5 -95.2) of incident MDR-TB cases in previously treated individuals. [START_REF] Kendall | Burden of transmitted multidrug resistance in epidemics of tuberculosis: a transmission modelling analysis[END_REF]

Current treatment for drug-resistant tuberculosis

The WHO has issued multiple documents to guide the treatment of drug-resistant tuberculosis in the recent years. However, the main structure of what is known as the "conventional" treatment regimen has remained mostly unchanged. MDR-TB treatment should include 1) a late-generation fluoroquinolone, 2) a second-line injectable, 3) other firstand second-line drugs to reach a minimum of four likely effective drugs. In some versions of WHO recommendations, it was advised to add pyrazinamide to all treatment regimens, while in others the fifth drug could have been another one. The duration of treatment is of approximately 18 to 24 months, composed by an intensive phase for 6-9 months and a continuation phase up to the end of treatment. The second-line injectable drug is to be stopped at the end of the intensive phase, unless this drug is needed to reach the minimum of four effective drugs (i.e. in cases with advanced drug resistance patterns or intolerance to other drugs).

The efficacy of the conventional MDR-TB treatment is overall unacceptably low, while varying significantly according to the number of drugs to which the strain is resistant in addition to rifampicin. According to four large meta-analyses, favourable treatment outcomes are achieved in 54 to 62% of MDR-TB patients. [START_REF] Bastos | An updated systematic review and meta-analysis for treatment of multidrug-resistant tuberculosis[END_REF][START_REF] Orenstein | Treatment outcomes among patients with multidrug-resistant tuberculosis: systematic review and meta-analysis[END_REF][START_REF] Ahuja | Multidrug Resistant Pulmonary Tuberculosis Treatment Regimens and Patient Outcomes: An Individual Patient Data Meta-analysis of 9,153 Patients[END_REF][START_REF] Johnston | Treatment outcomes of multidrug-resistant tuberculosis: a systematic review and meta-analysis[END_REF] Among XDR-TB patients, the rate of favourable outcomes decreased to 20% and 44% in two meta-analyses. [START_REF] Bastos | An updated systematic review and meta-analysis for treatment of multidrug-resistant tuberculosis[END_REF][START_REF] Jacobson | Treatment Outcomes among Patients with Extensively Drug-Resistant Tuberculosis: Systematic Review and Meta-Analysis[END_REF] In particular, the additional resistance to any fluoroquinolone appears to have the biggest impact on MDR-TB treatment outcomes [START_REF] Falzon | Resistance to fluoroquinolones and second-line injectable drugs: impact on multidrug-resistant TB outcomes[END_REF][START_REF] Kim | Treatment Outcomes and Survival Based on Drug Resistance Patterns in Multidrug-resistant Tuberculosis[END_REF][START_REF] Bonnet | Identification of patients who could benefit from bedaquiline or delamanid: a multisite MDR-TB cohort study[END_REF] and on overall treatment costs for the health system. [START_REF] Diel | Costs of tuberculosis disease in the European Union: a systematic analysis and cost calculation[END_REF][START_REF] Günther | Availability, price and affordability of anti-tuberculosis drugs in Europe: a TBNET survey[END_REF] In addition, among patients whose disease is not cured, mortality rates during long-term follow-up are high, in particular for XDR-TB and in HIV-infected patients. [START_REF] Gelmanova | Case fatality among patients who failed multidrug-resistant tuberculosis treatment in Tomsk, Russia[END_REF][START_REF] Pietersen | Long-term outcomes of patients with extensively drug-resistant tuberculosis in South Africa: a cohort study[END_REF] Patients with failed treatment are often discharged in the community, posing serious challenges to infection control and leading to increased transmission of the disease. [START_REF] Dheda | Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study[END_REF][START_REF] Shah | Transmission of Extensively Drug-Resistant Tuberculosis in South Africa[END_REF] In France, no routine data assessing treatment outcomes of M/XDR-TB cases are available at a national level. The notification system has recently included information on treatment outcome of tuberculosis cases, but it collects data only up to one year after the beginning of treatment and is therefore unable to capture outcomes of drug-resistant cases. [START_REF] Antoine | Treatment outcome monitoring of pulmonary tuberculosis cases notified in France in 2009[END_REF] The CNR-MyRMA has performed two studies to assess the treatment of M/XDR-TB cases in

France. The first one has showed that the favourable outcome rate of MDR-TB in 1994 was only 41%. [START_REF] Flament-Saillour | Outcome of multi-drugresistant tuberculosis in France: a nationwide case-control study[END_REF] Subsequently, an intervention program including targeted training and support to treating clinicians was implemented, which led to an increase of favourable outcomes up to 70% in 1998-1999. [START_REF] Uffredi | An intervention programme for the management of multidrugresistant tuberculosis in France[END_REF] In the following years, no nationwide survey of treatment outcomes of drug-resistant tuberculosis has been performed.

The conventional MDR-TB treatment is burdened by toxicity that occurs nearly universally in patients receiving standard-of-care treatment for MDR-TB. Common adverse events include: ototoxicity, liver and renal toxicity, gastrointestinal disorders, electrolyte imbalance, hypothyroidism, and neurotoxicity. Safety results in the control arm of the pivotal clinical trials of two drugs, bedaquiline and delamanid, recently approved by stringent regulatory authorities for MDR-TB treatment, are emblematic in this regard. In the bedaquiline C208 Stage 2 study, 98% of participants experienced adverse events; 36% had a severe adverse event and 6% had to discontinue treatment. [START_REF] Diacon | Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline[END_REF] In the delamanid Trial 204 study, 94% of participants experienced adverse events after only 8 weeks of treatment, 9% of them severe. [START_REF] Gler | Delamanid for multidrug-resistant pulmonary tuberculosis[END_REF] Observational cohorts corroborate the high frequency of side effects, with overall estimates ranging from 73% to 79%. [START_REF] Gelmanova | Case fatality among patients who failed multidrug-resistant tuberculosis treatment in Tomsk, Russia[END_REF][START_REF] Kvasnovsky | Treatment Outcomes for Patients with Extensively Drug-Resistant Tuberculosis, KwaZulu-Natal and Eastern Cape Provinces, South Africa[END_REF] This constellation of adverse events complicates the management of MDR-TB treatment: it has been shown that treatment-related adverse events are one of the major drivers of treatment default. [START_REF] Nathanson | Adverse events in the treatment of multidrug-resistant tuberculosis: results from the DOTS-Plus initiative[END_REF] In addition, the conventional treatment is poorly tolerated because it usually requires daily, painful intramuscular injections together with a large number of pills. This problem is even more pronounced for patients affected by comorbidities requiring complex treatment, like HIV infection or diabetes mellitus, or in children because of the absence of paediatric formulations.

Needs and priorities for research in drug-resistant tuberculosis treatment

The currently recommended treatment for M/XDR-TB is ineffective in an unacceptable portion of patients and burdened by potentially irreversible adverse events. This treatment regimen comprises painful daily injections and a large pill burden. The duration of almost two years inevitably reduces the treatment adherence and partially explains the large proportion of patients who default during treatment. In addition, some of the second-line antituberculosis drugs have additive toxicity and/or interactions with other drugs used to treat frequent comorbidities, like antiretrovirals. This desperate situation is due, in part, to an almost 50-year long drought in antituberculosis drug development. Recently, two new anti-tuberculosis drugs, bedaquiline and delamanid, have been finally approved for the treatment of MDR-TB. However, the pivotal trials that led to the approval of these drugs simply added each to a background regimen, retaining the toxic, long, conventional regimen. Without incorporating these newly approved drugs into novel regimens, their impact on the burden of disease will be limited. There is therefore dire need for large, sufficiently powered, controlled Phase III clinical trials testing new combinations of anti-tuberculosis drugs including new and re-purposed drugs in shorter treatment regimens.

According to a recent position paper, the main criteria that should be met by such a treatment to configure a real break-through in M/XDR-TB treatment are the following: [START_REF] Brigden | Principles for designing future regimens for multidrug-resistant tuberculosis[END_REF] • Contain at least one new class of drug;

• Include regimens for MDR-and XDR-TB strains;

• Contain three to five effective drugs, each from a different drug class;

• Be delivered orally;

• Have a simple dosing schedule;

• Have a good side-effect profile that allows limited monitoring;

• Last a maximum of 6 to 9 months;

• Have minimal interaction with antiretroviral drugs. However, results from such trials are year away from being available to impact treatment recommendations for M/XDR-TB. It is therefore equally important to perform operational research, in particular with carefully conducted prospective observational studies, to provide timely evidence on the use of new combinations of anti-tuberculosis drugs. Phase IV pharmacovigilance studies will also be of paramount importance in the following years to complement our current knowledge of the safety of new and re-purposed drugs. In red the new antimycobacterials, bedaquiline and delamanid. Re-purposed drugs (linezolid, clofazimine, meropenem, and imipenem) are also included in the figure. Source: Olaru et al. [START_REF] Olaru | Novel drugs against tuberculosis: a clinician's perspective[END_REF]

III. New anti-tuberculosis treatment

Re-purposed anti-tuberculosis drugs

Carbapenems

Carbapenems, including the association of imipenem and cilastatin, meropenem, ertapenem, and others, are a family of antibiotics belonging to beta-lactams that are commonly used to treat infections by drug-resistant Gram-negative bacteria. Beta-lactams exert their bactericidal effect by inhibiting the cell wall synthesis (Figure 7). Initially considered as not effective against M. tuberculosis, carbapenems had sparked new interest as anti-tuberculosis drugs after the discovery that they are effective when used together with beta-lactamase inhibitors such as amoxicillin-clavulanate. [START_REF] Hugonnet | Meropenem-Clavulanate Is Effective Against Extensively Drug-Resistant Mycobacterium tuberculosis[END_REF] In vitro studies have defined MICs of M. tuberculosis strains for meropenem/clavulanate at 0.125 to 2 mg/L 73 and confirmed the synergy of carbapenems and amoxicillin/clavulanate. [START_REF] Gonzalo | Is there a place for -lactams in the treatment of multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between meropenem and amoxicillin/clavulanate[END_REF] In the mouse model, both the combinations of imipenem/cilastatin and meropenem with clavulanate have shown anti-mycobacterial activity, although weaker than isoniazid. [START_REF] Veziris | Activity of Carbapenems Combined with Clavulanate against Murine Tuberculosis[END_REF] A few studies provide clinical evidence on the use of carbapenems as part of M/XDR-TB treatment regimens. A retrospective cohort showed good safety and satisfactory treatment outcomes in 37 difficult-to-treat MDR-TB patients treated in Italy. [START_REF] De Lorenzo | Efficacy and safety of meropenem-clavulanate added to linezolid-containing regimens in the treatment of MDR-/XDR-TB[END_REF] These results were confirmed by a larger, multinational cohort comparing 96 patients treated with meropenem/clavulanate to 168 who were treated with a meropenem/clavulanate-sparing regimen (n=168), and not showing major differences in treatment outcome or toxicity. [START_REF] Tiberi | Effectiveness and safety of meropenem/clavulanate-containing regimens in the treatment of MDR-and XDR-TB[END_REF] The same group of meropenem/clavulanate-treated patients was compared to a group of 84 patients receiving imipenem and cilastatin plus clavulanate, reporting a higher efficacy in the meropenem group. [START_REF] Tiberi | Comparison of effectiveness and safety of imipenem/clavulanateversus meropenem/clavulanate-containing regimens in the treatment of MDR-and XDR-TB[END_REF] In addition, a recent Phase IIa 14-day EBA clinical trial has shown that the administration of meropenem/clavulanate, given intravenously thrice daily, exerts a bactericidal activity on M. tuberculosis, although significantly lower than that of the first-line anti-tuberculosis treatment. [START_REF] Diacon | β-Lactams against Tuberculosis -New Trick for an Old Dog?[END_REF] The main challenge in the implementation of carbapenems is the need of intravenous twice (imipenem-cilastatin) or thrice (meropenem) daily administration which, together with the elevated costs, make these drugs challenging to use in low-resource settings and for outpatients. For these reasons, ertapenem, a carbapenem which requires only once daily parenteral administration, has been considered an ideal candidate to integrate the antituberculosis armamentarium. However, most studies assessing the efficacy of ertapenem in vitro or in animals have provided disappointing results. [START_REF] Veziris | Activity of Carbapenems Combined with Clavulanate against Murine Tuberculosis[END_REF] In contrast, a recent hollow-fibre model-based study has suggested that ertapenem might have a good bactericidal and sterilising efficacy. [START_REF] Van Rijn | Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis and Implications on Clinical Dosing[END_REF] Notwithstanding the availability of a few case reports of ertapenemtreated patients, 81 the use of this drug is still not supported by enough evidence to be considered in routine anti-tuberculosis treatment.

Overall, the evidence supporting the use of carbapenems is still limited, and mainly relying on a Phase II EBA study. The observational studies which were cited before have all multiple methodological weaknesses which complicate their interpretation: namely, the evaluation of treatment efficacy is confounded by the other drugs of the regimen, the duration of administration and timing of introduction of each of them, and additional interventions like surgery. As it often is the case in retrospective observational studies, the collection of safety data was not systematic nor standardised and is therefore of little value.

Clofazimine

Clofazimine is a riminophenazine that is active against most mycobacteria but has been prevalently used for the treatment of leprosy. Clofazimine's mechanism of action has not been completely elucidated but appears to be linked to the destabilization of the mycobacterial cell wall (Figure 7). Initially discarded as an anti-tuberculosis drug for excessive toxicity, clofazimine's role has been recently re-assessed following the reduction of its dose from 400 mg daily to the actually recommended 100 mg daily, which has led to a better tolerability.

The activity of clofazimine appears to be independent of the administered dose, supporting the reduction of its posology. [START_REF] Swanson | Pharmacokinetics and Pharmacodynamics of Clofazimine in a Mouse Model of Tuberculosis[END_REF] In the mouse model, clofazimine has shown to have a sterilizing activity against M. tuberculosis in both first-and second-line anti-tuberculosis treatment regimens. [START_REF] Tyagi | Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis[END_REF][START_REF] Grosset | Assessment of Clofazimine Activity in a Second-Line Regimen for Tuberculosis in Mice[END_REF] In addition, clofazimine has shown to exert a prolonged activity after its interruption, due probably to its long half-life, a property that may make clofazimine an important component of shorter treatment regimens. [START_REF] Swanson | Clofazimine Contributes Sustained Antimicrobial Activity after Treatment Cessation in a Mouse Model of Tuberculosis Chemotherapy[END_REF] Observational studies have renewed the interest in clofazimine. In particular, clofazimine is a component of the WHO-approved shorter (9 to 12 months) MDR-TB regimen that has been first developed in Bangladesh and then in other Western and Central African countries, showing exciting results. [START_REF] Aung | Successful '9-month Bangladesh regimen' for multidrug-resistant tuberculosis among over 500 consecutive patients[END_REF][START_REF] Kuaban | High effectiveness of a 12-month regimen for MDR-TB patients in Cameroon[END_REF][START_REF] Piubello | High cure rate with standardised short-course multidrug-resistant tuberculosis treatment in Niger: no relapses[END_REF][START_REF] Trébucq | Treatment outcome with a short multidrug-resistant tuberculosis regimen in nine African countries[END_REF][START_REF] Van Deun | Highly Effective, and Inexpensive Standardized Treatment of Multidrug-resistant Tuberculosis[END_REF] Systematic reviews and meta-analyses of clofazimine for MDR-TB have reported pooled success rates similar to those generally reported for MDR-TB treatment, despite the fact that clofazimine has been often reserved for patients with few treatment options: in two studies; clofazimine-receiving patients achieved a favourable treatment outcome rate of 62% (95% CI: 53%-71%) [START_REF] Dey | Outcomes of clofazimine for the treatment of drug-resistant tuberculosis: a systematic review and meta-analysis[END_REF] and 65% (95% CI: 54%-76%), respectively. [START_REF] Gopal | Systematic review of clofazimine for the treatment of drug-resistant tuberculosis[END_REF] In a retrospective cohort including XDR-TB patients with advanced resistance patterns, the use of clofazimine was associated with improved culture conversion rates. [START_REF] Padayatchi | Clofazimine in the treatment of extensively drug-resistant tuberculosis with HIV coinfection in South Africa: a retrospective cohort study[END_REF] A recent cohort study has showed an overall reassuring safety profile and a total favourable outcome rate of 60.9% in 1446 MDR-TB patients treated with clofazimine in Brazil. [START_REF] Dalcolmo | Effectiveness and safety of clofazimine in multidrug-resistant tuberculosis: a nationwide report from Brazil[END_REF] Limited clinical trial evidence is available for clofazimine. In a recent Phase IIa EBA study, clofazimine had no measurable activity alone or when added to a combination with bedaquiline, pretomanid and/or pyrazinamide at 14 days in 15 treatment-naïve, sputum smearpositive patients with pulmonary TB. A regimen containing pretomanid, pyrazinamide, and clofazimine, however, had activity that was not distinguishable from neither the most active experimental regimen nor from the standard first-line anti-tuberculosis treatment. [START_REF] Diacon | Bactericidal Activity of Pyrazinamide and Clofazimine Alone and in Combinations with Pretomanid and Bedaquiline[END_REF] These findings are similar to those of a 14-day EBA study performed both in vitro and in the mouse model, showing only weak and late activity of clofazimine, with no clear relationship with clofazimine dose. [START_REF] Ammerman | Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo[END_REF] Results are also available from a randomized, controlled clinical trial including 105 MDR-TB patients, which compared outcomes among participants receiving clofazimine plus background regimen (n=53) and participants receiving only background regimen without clofazimine (n=52). Treatment success was reported in 76% of the clofazimine group and 54% in the control group (p=0.035). Furthermore, interim favourable endpoints assessed by the study (cavity closure and accelerated sputum culture conversion) occurred more frequently in the clofazimine arm. [START_REF] Tang | Clofazimine for the Treatment of Multidrug-Resistant Tuberculosis: Prospective, Multicenter, Randomized Controlled Study in China[END_REF] Overall, the use of clofazimine to treat M. tuberculosis infection is supported more by observational data than by interventional studies. Phase II EBA trials did not find any early bactericidal efficacy, while the Phase III clinical trial has been criticized for the unclear design and the absence of a reported sample size calculation. Conversely, many prospective cohorts have confirmed the role of clofazimine in the WHO-recommended shorter regimen.

Clofazimine appears to have an excellent safety profile, which is confirmed by data coming from the prolonged use as an anti-leprosy drug.

Linezolid

Linezolid is a component of the antibiotic class of oxazolidinones, which acts by inhibiting the ribosomal protein synthesis (Figure 7). Linezolid has been developed and is indicated to treat bacterial infections from drug-resistant Gram-positive agents. In the last years, it has been re-purposed for tuberculosis treatment. Linezolid has demonstrated activity against laboratory and clinical strains of pan-susceptible and drug-resistant M. tuberculosis. 98- 100 In addition, multiple studies have illustrated the bactericidal and sterilizing activity of linezolid and other oxazolidinones, such as sutezolid, as part of multi-drug regimens in the mouse model. [101][102][103][104] There is growing evidence from clinical studies on the efficacy of linezolid against M.

tuberculosis. Multiple MDR-TB case series have reported relatively high rates of treatment success in difficult-to-treat patients using linezolid. [105][106][107][108] Four systematic reviews and metaanalyses have studied linezolid-containing regimens for the treatment of MDR-TB patients: the first showed a pooled proportion of treatment success of 68% (95% CI: 58.0%-78.99%), 109 the second showed a 93.5% MDR-TB sputum culture conversion rate and 81.8% successful treatment outcome, 110 the third reported a pooled treatment success rate of 77%, 111 and the fourth a favourable outcome rate of 83% (95% CI: 75-90%). 112 High-quality evidence supporting the use of linezolid comes from two clinical trials.

In the first, performed in South Korea, linezolid was added to failing XDR-TB regimens: patients were randomly assigned to immediate or delayed (after 2 months) linezolid therapy. By 4 months, 79% (15/19) of patients in the immediate-start group, compared with 35% (7/20) of patients in the delayed-start group, had culture conversion (p=0.001). Overall, 87% of patients (34/39) had a negative sputum culture within 6 months after linezolid had been added to their drug regimen. It is worth noting that, despite the fact that linezolid was added as a single drug to failing regimens, failure was relatively infrequent (11% [4/38]). 113 Overall, 27 patients out of 38 (71%) with chronic XDR-TB were declared cured at the end of treatment, and no relapse was detected up to 12 months post-treatment. 114 In the second trial, performed in China, 65 patients receiving multidrug treatment for XDR-TB were randomly assigned to linezolid or a control. The treatment success rate in the linezolid group was 69%, significantly higher than that in the control group (34%, p=0.004). 115 Overall, the key role of linezolid in the treatment of M/XDR-TB appears to be established. Although the results of the two clinical trials supporting its clinical efficacy are partially discussable due to some controversial design choices, there is a remarkable amount of additional observational evidence. The main drawback of this drug appears to be the frequent, potentially irreversible toxicity, which is associated to its long-term use. The articles were retrieved with the following research strategies: "bedaquiline" AND "tuberculosis" plus "TMC207" AND "tuberculosis" (blue), "delamanid" AND "tuberculosis" (orange), "new drugs" AND "tuberculosis" (grey).

*The search for the year 2018 was censored on June 30th, 2018.

Methodology and results of evidence search

The objective of the literature search was to identify all available evidence on the new anti-tuberculosis drugs bedaquiline and delamanid, focusing in particular on the following aspects:

•

Drug structure The following research terms were used: "bedaquiline" AND "tuberculosis", "TMC207" AND "tuberculosis", "delamanid" AND "tuberculosis", "new drugs" AND "tuberculosis". Abstracts and titles were systematically screened: for studies containing original research related to the above topics, full manuscripts were acquired and reviewed.

Overall, 11 review articles [116][117][118][119][120][121][122][123][124][125][126] and 4 systematic reviews [127][128][129][130] were identified: these articles were reviewed to consolidate the comprehensiveness of the literature search.

Figure 8 shows the number of abstracts identified per year with the research strategy described above.

Bedaquiline

Structure and mechanism of action

Bedaquiline represents the first compound of a novel class of anti-mycobacterial drugs, the diarylquinolines. Bedaquiline was previously known as TMC207 and R207910

during its development and is currently marketed with the commercial name of Sirturo ® by Janssen Pharmaceuticals (Belgium). Bedaquiline is the most promising among the diarylquinolines that have been identified through systematic testing of approximately 70 000 compounds on strains of M. smegmatis, which represents an easier target for drug development than M. tuberculosis. 131 The conformation of bedaquiline has been characterised combining magnetic resonance imaging with molecular modelling, 132 and subsequently confirmed using X-ray diffraction. 133 These studies allowed to define the specific structure of the quinoline nucleus of the diarylquinolines, characterised by the functionalized lateral (3') chain, which differentiates them from other molecules containing a quinoline group. 122 Figure 9 shows the structure and absolute conformation of bedaquiline, defined chemically as 1-(6bromo-2-methoxy-quinolin-3-yl)-4-dimethylamino-2-naphthalen-1-yl-1-phenyl-butan-2-ol, or with the general formula C32H31BrN2O2; its molecular weight is 555.51 Da. 131 The molecular target of bedaquiline is an ATP synthase, a very well-conserved membrane protein that plays a fundamental role in the energetic metabolism and is ubiquitarian across prokaryotes and eukaryotes. The mycobacterial ATP synthase is composed by a transmembrane and a cytoplasmic part, each constituted by different subunits (Figure 10). It is essential for the survival of mycobacteria, regardless of their metabolic status and environment: actively replicating and dormant, intra-and extracellular. 134 Bedaquiline affinity for the mycobacterial ATP synthase is much higher than the one for human ATP synthase, a

reassuring finding in view of potential toxicity concerns. 135 The mutations conferring resistance to bedaquiline were identified through whole genome sequencing of resistant M.

tuberculosis isolates selected in vitro and were all concentrated in the atpE gene. This gene codes for the c subunit of the transmembrane component (called F0) of the ATP synthase. 136 Bedaquiline binds the c subunit at the level of amino acids 28, 59, 61, 63, and 66: 137 Docking studies have shown that bedaquiline has high affinity for the ATP synthase even at low proton motive force and with low pH values. 138 The binding of bedaquiline to the ATP synthase results in the disruption of the normal functioning of the chain of protonic transfer. 139 This alteration of the subunit c interface of the F0 component of the ATP synthase leads to futile proton cycling and consequently to death of mycobacteria. 140 In addition, it has been shown that bedaquiline also binds to the ε subunit of the F0 component of the ATP synthase. 141 This finding has been further confirmed by showing that the replacement of an amino acid of the chain of ε subunit resulted in bacterial hypersusceptibility to bedaquiline. 142

Pharmacokinetics

In the mouse model, the oral administration of bedaquiline leads to a prolonged diffusion of the molecule in the tissues and to a long half-life in both plasma (range: 43.7-64.0 h) and tissues (range: 28.1-92.0 h). 131 In humans, the half-life is of approximately 24 h and the terminal half-life is extremely long, up to 4-5 months: this is most likely due to redistribution from the tissue compartments. 143 The bioavailability of bedaquiline after oral administration is high; the administration with food (standard meal) results in a significant increase in absorption compared with fasting conditions. The peak of plasmatic concentration (CMAX) is reached after a median of 5 h following drug administration. Both CMAX and the area under the curve (AUC) of plasmatic concentrations increase proportionally to the increase of the administered dose of bedaquiline. 131 Overall, bedaquiline activity seems to be principally linked to the AUC and therefore to the cumulative weekly dose, rather than to the frequency of administration: the pharmacokinetic profile seems therefore favourable for intermittent administration of the drug. 144 In a Phase II clinical trial evaluating the early bactericidal activity (EBA) of 400 mg daily bedaquiline, the CMAX reached 5.5 mg/L and the 24-hour AUC was 64,75 mg/h/L. 145 Bedaquiline is metabolised by the liver to a N-desmethyl derivate (M2) that has a residual bactericidal activity, although 5 times weaker than bedaquiline, and then further metabolised to two derivates that have almost no residual activity (M3 and M4). Both bedaquiline and its metabolites have very high levels (>99.9%) of binding with plasmatic proteins. Metabolism is mainly mediated by the CYP3A4 isoenzyme of P450 cytochrome, and less importantly by CYP2C8 and CYP2C19. 146 As a consequence, bedaquiline should be used with caution in patients with mild or severe hepatic impairment; conversely, the drug can be used safely in patients with renal impairment. Elimination is mainly through faeces: the renal clearance is negligible.

Bedaquiline is a lipophilic compound, with good penetration into body tissues such as the lungs. In a study on the C3HeB/FeJ mouse model, which has the advantage of closely mimic the pathophysiological conditions of human lungs as it develops caseous necrotic granulomas, bedaquiline was shown to accumulate more in the highly cellular regions of the lungs, and at a lower but still biologically relevant concentration within the central caseum. 147 Moreover, in mice, it was shown with liquid chromatography / tandem mass spectrometry that bedaquiline administration led to wide distribution into the brain, reaching the CMAX in 4 hours after administration. 148 In humans, a similar technique combining liquid chromatography with tandem mass spectrometry has been validated for the measurements of serum levels of bedaquiline. 149 In a case of a MDR-TB patient with tuberculous meningitis who was receiving a bedaquiline-containing regimen, this method showed therapeutic bedaquiline concentrations in the serum, but undetectable levels in the cerebrospinal fluid. 150 This finding has however been criticised with regard to the collection methods that were used and that may not be the most suitable for a highly-lipophilic compound such as bedaquiline. 151

Drug-drug interactions

The P450-mediated liver metabolism makes bedaquiline prone to drug-drug interactions with other molecules that inhibit or induce the P450 cytochrome activity. The interaction of bedaquiline with rifamycins have been relatively well studied. The coadministration of rifampicin was associated in the mouse model to a 50% reduction of plasmatic concentrations of bedaquiline, although this did not affect the treatment efficacy. 153 These findings were confirmed in healthy volunteers, where rifampin reduced bedaquiline AUC by approximately 45%. In the same study, rifabutin, which is also part of the rifamycin family but usually less prone to drug interactions, resulted in little quantitative impact on bedaquiline exposure but was associated with severe (Grade 3 and 4) adverse events before and after the end of the 29-day bedaquiline treatment course. 154 Consequently, the coadministration of any rifamycin and bedaquiline is contraindicated. More generally, the coadministration of bedaquiline with any moderate or strong inducer or inhibitor of the cytochrome P450 is likely to have relevant pharmacokinetic consequences and should be avoided if possible (see Table 1). The pharmacokinetic interaction between bedaquiline and other second-line antituberculosis has not been studied into detail, except for the co-administration of clofazimine that was not found to lead to a statistically significant effect on bedaquiline concentrations. 159

Pre-clinical studies: in vitro

The spectrum of activity of bedaquiline is almost completely restricted to mycobacteria. In the first study describing the in vitro activity of this drug, the minimum inhibitory concentrations (MIC) of bedaquiline on M. tuberculosis were between 0.03 and 0.12 mg/L. As expected, in light of the novel mechanism of action compared to the other antituberculosis compounds, the same MICs were reported on MDR M. tuberculosis strains. 131 Another studies analysed a significant number of drug-susceptible and MDR M. tuberculosis strains and identified the median MIC at 0.03 mg/L. 160 Remarkably, bedaquiline was shown to be equally effective in vitro against dormant mycobacteria as against actively replicating ones. 136 This finding was replicated in a dormancy model where bedaquiline and other lipophilic drugs were more active than hydrophilic agents against dormant M. tuberculosis strains in hypoxic conditions at low pH. 161 The in vitro bactericidal action mediated by bedaquiline seems to be more effective on intracellular mycobacteria. 162 Several studies have assessed the synergy of bedaquiline with other antimycobacterials and with other drugs. In whole blood cultures of healthy volunteers, the combination of rifabutin plus bedaquiline produced enhanced intracellular mycobactericidal activity compared the sum of their individual effects. This synergy not found between bedaquiline and rifampicin. 163 Another study on whole blood cultures testing different drug associations against M. tuberculosis suggested that the most active drug combinations were those containing bedaquiline plus sutezolid (a component of the oxazolidinone family, like linezolid) or SQ109 (a ethylenediamine derived from ethambutol), two promising compounds currently being developed, which were predicted to have cumulative activity comparable to standard first-line anti-tuberculosis treatment. In this study, pretomanid (a nitroimidazole like delamanid) and bedaquiline interacted antagonistically. 164 The co-administration of SQ109 increases the bacterial killing and post-antibiotic effect of bedaquiline, and reduces its MIC. 165 Similar findings have been reported for the association of bedaquiline and BTZ043 (a benzothiazinone). 166 The combination of linezolid and bedaquiline has also shown an additive bactericidal activity against both dormant and actively replicating populations. 167 In a recent study, the administration of verapamil, a calcium channel blocker and an inhibitor of mycobacterial efflux pumps, was shown to potentiate the in vitro activity of bedaquiline. However, this was not the consequence of increased intrabacterial bedaquiline, as expected, but rather the result of verapamil's ability to directly impact membrane energetics 168 and to enhance systemic exposure to companion drugs via effects on mammalian transporters. 169 Bedaquiline appears as a promising candidate as part of treatment regimens for other non-tuberculous mycobacteria, such as M. ulcerans, M. abscessus, M. fortuitum, M. kansasii, M. marinum, and the mycobacteria belonging to the M. avium complex (MAC). 131,160,170 Other species, like M. xenopi, appear to be naturally resistant to this drug. 171 However, a detailed summary of the efficacy of bedaquiline on non-tuberculous mycobacteria is outside of the scope of this work.

Pre-clinical studies: animal model

There is a rich amount of evidence on bedaquiline activity coming from pre-clinical studies performed in the animal model and in particular in mice. These studies provided the first in vivo data on bedaquiline. In the non-established TB infection mouse model, bedaquiline showed a bactericidal activity on M. tuberculosis starting from a minimum posology of 12.5 mg/kg 4 times weekly. The addition of bedaquiline to the standard first-line anti-tuberculosis regimen led to a significant increase of the bactericidal efficacy. 131 Similar results were obtained by studies testing the activity of bedaquiline in guinea pigs. 172,173 A primary objective of studies in mice is testing the activity of bedaquiline in combination with other antimycobacterials. This has been done using different protocols in the established TB infection mouse model in order to test bactericidal and sterilising activity.

The combination of bedaquiline and pyrazinamide was shown to exert a synergistic bactericidal activity in mice, compared to other combinations including isoniazid, rifampicin, and moxifloxacin. 174 Moreover, this combination has also demonstrated remarkable sterilising capabilities. 175 These results support the findings that combinations including bedaquiline and pyrazinamide might have the potential to shorten the treatment of drug-susceptible tuberculosis from 6 to 4 months. 176 Similarly, once-weekly administration of bedaquiline, in combination with pyrazinamide and rifapentine, appears to have a sterilising efficacy that exceeds the one of the first-line daily anti-tuberculosis regimen. 177 Bedaquiline has also been tested in association with second-line drugs. The combination of bedaquiline plus amikacin, moxifloxacin, pyrazinamide, and ethionamide, allowed to sterilize the culture of mice lungs after only two months of treatment (bactericidal activity) 178 and to prevent the appearance of relapse 6 months after the end of treatment (sterilising activity). 179 Moreover, drug combinations including only new or experimental drugs, such as bedaquiline plus clofazimine, sutezolid, and/or pretomanid, with or without pyrazinamide, were proven to exert both bactericidal and sterilising efficacy on M. tuberculosis in mice, with the potential to surpass the efficacy of current standard first-line treatment. 180 These findings were replicated using the combination of bedaquiline, pretomanid, and linezolid, 181 and the association of bedaquiline with sutezolid and a nitroimidazole between pretomanid and TBA-354, with or without pyrazinamide. 182 In a recent study, the combination of bedaquiline plus pretomanid, moxifloxacin and pyrazinamide, rendered all mice relapse-free after only two months of treatment. In addition, data provided by this study seemed to suggest that pyrazinamide could be discontinued after the first month of treatment without compromising the sterilising activity of the drug combination. 183

Among non-antimycobacterials, bedaquiline has been tested in combination with verapamil in two studies, showing that the coadministration of verapamil with subinhibitory doses of bedaquiline gave the same bactericidal effect in mice as did the full human bioequivalent bedaquiline dosing. 169,184 In contrast with what was expected, the administration of verapamil had no effect on clofazimine activity. 169 One study has assessed in mice the interest of bedaquiline in the treatment of latent tuberculosis infection: a monotherapy with bedaquiline for 3 to 4 months may be a promising alternative to current standard treatment. 185

Among non-tuberculous mycobacteria, studies in mice have shown that bedaquiline has bactericidal activity against M. leprae 186 and M. ulcerans, 170 bacteriostatic activity against M. avium, 187 and almost no activity on M. abscessus. 188 In two EBA studies, bedaquiline was tested alone against different comparators. In the first study, the 7-day EBA of bedaquiline at different posologies (25 to 400 mg daily) was compared to the one of rifampicin and isoniazid: significant bactericidal activity of 400 mg daily of bedaquiline was observed from day four of administration and was similar in magnitude to comparators over the same period, although lower overall (Figure 11). These findings suggest that bedaquiline exerts a late-onset bactericidal activity with a clear relationship between dose and response. 189 In the second study, the 14-day EBA of standard first-line anti-tuberculosis treatment was compared to the one of bedaquiline given at daily doses of 100 mg, 200 mg, 300 mg, and 400 mg, preceded by loading doses (200 mg, 400 mg, 500 mg, and 700 mg on the first treatment day, and 100 mg, 300 mg, 400 mg, and 500 mg on the second treatment day, respectively). The EBA in the 400 mg dose group was greater than that in the 100 mg dose group, although significantly inferior to the one of the first-line treatment association. All the bedaquiline groups showed significant delayed-onset bactericidal activity that was continued to the end of the 14-day evaluation period (Figure 12).

The results of this dose-ranging study confirmed the existence of a linear dose/efficacy trend and supported the use of a loading dose for bedaquiline. 190

Kaplan-Mayer curve showing the time to culture conversion of sputum samples of MDR-TB patients receiving either bedaquiline (TMC207) or placebo plus optimised background regimen in study C208

Stage 1. Source: Diacon et al. 192 In the other two EBA studies, bedaquiline was tested as part of multidrug regimens.

One study tested the 14-day EBA of parallel groups receiving 1) bedaquiline, 2) bedaquilinepyrazinamide, 3) PA-824-pyrazinamide, 4) bedaquiline-PA-824, 5) PA-824-moxifloxacinpyrazinamide, and 6) standard antituberculosis treatment. The bedaquiline-containing regimens showed a good EBA, although in average lower compared to PA-824 (pretomanid)based regimens. Among bedaquiline-containing arms, the highest EBA was exerted by the combination of bedaquiline and pyrazinamide, confirming the findings of existing synergy between these two drugs. 193 Another, more recent study had a similar approach assessing the 14-days EBA of seven treatment arms: 1) bedaquiline-pyrazinamide-clofazimine; 2) bedaquiline-pretomanid-pyrazinamide; 3) bedaquiline-pretomanid-pyrazinamide-clofazimine;

4) bedaquiline-pretomanid-clofazimine; 5) clofazimine alone; 6) pyrazinamide alone; 7) standard first-line treatment. The highest EBA was recorded in the bedaquiline-pretomanidpyrazinamide arm; this treatment association showed a significantly higher bactericidal activity than the standard first-line treatment. 194 The results of these EBA studies allowed characterising the bactericidal activity of bedaquiline, defining its posology, and identifying the most promising drug combinations to bring forward in subsequent clinical trials. However, the main sources of evidence, which led to the provisional approval of bedaquiline for MDR-TB treatment, are two Phase IIb studies, C208 Stage 1 and C208 Stage 2. C208 Stage I is a randomized, controlled trial, where 47 patients with pulmonary MDR-TB were randomly assigned to receive either bedaquiline (n=23) (400 mg daily for 2 weeks, followed by 200 mg three times a week for 6 weeks) or placebo (n=24) in combination with an optimised background regimen. The primary efficacy endpoint was the time to conversion of sputum cultures. The bedaquiline arm showed a significantly faster time to culture conversion compared to the placebo arm (hazard ratio 11.8, 95% confidence interval 2.3 to 61.3, p = 0.003) and a higher proportion of sputum culture conversion at 8 weeks from treatment start (48% vs. 9%) (Figure 13). 192 A subsequent study reported the results of 2-years follow-up of the participants of the C208 Stage 1 trial. The median time to culture conversion was 78 days in the bedaquiline arm and 129 days in the placebo arm, while culture conversion rates at 24 weeks were 81.0% and 65.2% in the bedaquiline and placebo arms, respectively. At 104 weeks, treatment success rates were 52.4% in the bedaquiline group and 47.8% in the placebo group. Resistance to companion drugs was acquired in only one patient receiving bedaquiline, compared to five patients with pulmonary, smear-positive MDR-TB were randomly assigned to receive either bedaquiline (n=79) (400 mg daily for 2 weeks, followed by 200 mg three times a week for 22 weeks) or placebo (n=81) in combination with an optimised background regimen. The primary endpoint was the time to sputum-culture conversion. Patients were followed for 120 weeks from baseline. The main analyses were performed on the 132 patients (66 bedaquiline arm, 66 placebo arm) belonging to the modified intention-to-treat population. Compared with the placebo arm, the bedaquiline arm showed shorter median time to culture conversion, 125 days vs 83 days (hazard ratio 2.44, 95% confidence interval 1.57 to 3.80) (Figure 14), and higher rates of culture conversion at 24 weeks (79% vs 58%, p=0.008) and at 120 weeks (62% vs 44%, p=0.04). At the end of treatment, the rate of cure according to WHO treatment outcomes was higher in the bedaquiline arm (58% vs 32%, p=0.003) (Figure 15). However, the very low rate of cure in the placebo arm (32%) should be noted and partially questions the value of the results. 66

Table 3. Characteristics of the study participants of the three bedaquiline Phase IIb trials.

Source: Diacon et al, Pym et al. [START_REF] Diacon | Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline[END_REF]192,195,196 Study The characteristics of the study population of the three Phase IIb trials are summarised in Table 3, while Table 4 shows their efficacy outcomes. Remarkably, all patients with any previous exposure to second-line drugs or harbouring XDR-TB strains were excluded from the two controlled trials. HIV-infection rates among study participants were low in all the trials. In addition, the optimised background regimens in studies C208 Stage 1 and Stage 2 did not include the most effective re-purposed drugs, linezolid and clofazimine.

Efficacy: observational studies

The first article describing the results of a Phase IIb clinical trial was published in 2009. Since then, a growing number of observational studies have been published describing case reports, case series, and cohorts of patients receiving bedaquiline as part of MDR-TB treatment regimens. Table 5 summarises the features of these studies. Studies discussing special populations (i.e. pediatric) or "off-label" use of bedaquiline (ie. administration beyond 24 weeks) will be presented into more detail later.

Overall, 23 observational studies were identified by the literature search. Most of them (14) were case reports or case series describing the efficacy of bedaquiline-containing regimens in less than 10 patients. Among the other nine studies, six mostly described interim efficacy outcomes, and only three described final (end-of-treatment) outcomes.

The three studies of this former group included a large multi-country retrospective cohort, a prospective cohort performed in South Africa, and a case series performed in India.

The first study (Borisov et al) described the treatment efficacy in 428 culture-confirmed MDR-TB cases receiving bedaquiline in 25 centres across 15 countries: sputum smear and culture conversion rates were 81.1% and 56.7% at 60 days, with a median (interquartile range) time to smear and culture conversion of 34 (30-60) days and 60 (33-90) days, respectively. End-of-treatment outcomes were known for 247 cases: 71.3% achieved success (62.4% cured; 8.9% completed treatment), 13.4% died, 7.3% defaulted and 7.7% failed. 197 In the second study (Olayanju et al), a cohort of 272 XDR-TB patients (49% HIV-infected) was prospectively followed from treatment start until outcome assignment. The authors compared treatment outcomes between patients who had not received bedaquiline (n=204), all treated before 2013, to those who had received bedaquiline (n=68), all treated since 2013, and among whom 80.9% also received linezolid. Favourable outcome rates were substantially higher (66.2% vs 13.2%, p<0.001) and rates of treatment failure were lower (5.9% versus 26.0%; p<0.001) among patients who had received bedaquiline. In addition, the mortality rate was also significantly lower in the bedaquiline group. 198 In the third study (Udwadia et al), end-oftreatment outcomes were available for 18 out of 20 MDR-TB patients: 11 were cured, 6 failed treatment, and one relapsed. 199 The six studies describing interim treatment efficacy outcomes reported culture conversion rates of MDR-TB regimens including bedaquiline ranging from 70.9% to 100% after 6 months of treatment. [200][201][202][203][204][205] Similarly, most of the case series and case reports reported overall satisfactory results of bedaquiline use. [206][207][208][209][210][211][212][213][214][215][216] A notable exception is a case report from Switzerland of a MDR-TB patient treated sequentially with bedaquiline and delamanid who failed treatment twice and developed resistance to both new drugs. This case report was the first description of the acquisition of cross-resistance between bedaquiline and clofazimine, mediated by a mutation in the gene Rv0678. [217][218][219] Table 6. Summary of main safety findings in the three bedaquiline Phase IIb trials.

Source: Diacon et al, Pym et al. [START_REF] Diacon | Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline[END_REF]192,195,196 Trial

Safety: clinical trials

The first Phase I studies testing the administration of increasing doses of bedaquiline in healthy volunteers showed a good safety profile of the drug. 131 Phase IIa bedaquiline EBA studies provided some limited evidence on the tolerability of the first seven to 14 days of bedaquiline administration. In the first EBA study, the incidence of adverse events was similar across all treatment groups. Two deaths occurred due to TB-related haemoptysis. 189 In the bedaquiline dose-ranging study, at least one adverse event considered as related to the study medication was reported in eight (13.3%) patients among those receiving bedaquiline and in two (25%) patients receiving the standard first-line treatment: these were mostly mild and mainly represented by headache, nausea, or vomiting.

One severe adverse event (SAE) was reported, an increase in liver enzymes in a patient receiving bedaquiline at 100 mg daily. 190 In the 14-day EBA multiagent study testing different combinations of bedaquiline with other new and conventional second-line drugs, adverse events were reported in more than half of patients, although most were mild and considered as not related to the investigational drugs. Seven patients were withdrawn due to adverse events: five with increased liver enzymes, one for QTcF increase, and one for altered consciousness due to newly diagnosed neurocysticercosis. 193 In the 14-day EBA study comparing combinations of bedaquiline with pretomanid, clofazimine, and/or pyrazinamide, adverse events were experienced by 61.9% of patients, of which 27.6% were these considered as treatment-related. Grade 3/4 adverse events were experienced by seven (6.7%) patients: three had increase in liver enzyme values, one increase in creatine kinase values, one case of decreased appetite, one case of gastroenteritis, and two cases of skin discoloration. Increase in mean QTcF values was found with all treatments, but no case of QTcF>500 ms was reported. 194 The main safety findings reported by the three Phase IIb clinical trials are summarised in Table 6. Taking together the two controlled trials (C208 Stage 1 and C208 Stage 2), the total rate of patients experiencing SAEs was higher among the bedaquiline arms than in the placebo arms (7% vs. 2%), although the rate of patients who had to discontinue any drug due to adverse events was comparable between the two groups. In particular, the rate of liver enzyme elevation was higher among patients treated with bedaquiline compared to placebo (9% vs. 2%), with two hepatic-related SAEs occurring in the bedaquiline arms. QT interval prolongation was also more pronounced in the bedaquiline arm than in the placebo arm (median QT increase: 15.7 ms vs. 6.2 ms). 220 In trial C209, the mean maximum change in QTcF interval from baseline was 14.2 ms and was significantly higher in patients concomitantly treated with clofazimine. 196 Remarkably; an unbalance in the overall rate of deaths has been reported in the two Stages of C208 trial: overall, 12% of participants died in the bedaquiline arms, compared to 4% in the placebo arms. 220 This difference is mainly driven by results of the C208 Stage 2 study. In this trial, 11 out of 12 of the deaths in the bedaquiline arm occurred after the end of treatment with bedaquiline, with a median delay of 425 days after the last bedaquiline dose. After a thorough analysis of the clinical history and circumstances of death of these cases, TB was considered as the main cause of death in half of patients in the bedaquiline arms. Among patients who died in the bedaquiline arm, none had QTcF interval prolongation of more than 60 ms during treatment, nor experienced QTcF values of 500 ms or more. [START_REF] Diacon | Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline[END_REF] In Study C209, there were 16 deaths (7% of total number of participants): 13 occurred after the end of bedaquiline treatment, four after withdrawal from the study, and one occurred in a HIV-positive patient. In nine cases, death was considered as related to TB or to a TB-related illness. No death was considered as related to bedaquiline. four patients after stopping delamanid and/or bedaquiline. 200 In a cohort of 28 patients treated with both bedaquiline and delamanid, no patient had an >500 ms QTcF interval values, while four had six instances of QTcF increase of >60 ms from baseline but none permanently discontinued the drugs. In addition, 16 SAEs were reported in seven patients. 201 In a cohort of children and adolescents receiving bedaquiline, five patients had grade 3 or 4 QTcF prolongation, and four patients experienced increases in QTcF >60 ms compared to baseline: however, no drug discontinuation was required. Recurrent prolongation of QTcF >500 ms was identified in one case and led to a change in treatment, but without stopping bedaquiline. 203 In a cohort of 91 patients from South Africa, one patient developed atrial fibrillation and subsequently discontinued this drug permanently; QTcF >500 ms values were reported in three patients. 204 In other cohort studies presenting interim efficacy results, safety data were not available. 202,205 In most case reports, the tolerability of bedaquiline was reported to be good and the treatment course with this drug could be completed. [207][208][209][210][211]214,215 In one case series of five MDR-TB patients receiving the combination of bedaquiline and delamanid, two developed a QTcF prolongation above 500 ms, without associated arrhythmias: however, none of the patients had to stop bedaquiline. 206 In another report, an Indian XDR-TB patient developed QTcF prolongation while receiving concomitantly bedaquiline and delamanid. Bedaquiline was discontinued temporarily and reintroduced after a few days. The patient did not experience any cardiac adverse event. 213 Other case reports did not present safety results of the treatment. In summary:

• Bedaquiline is a drug belonging to a drug class with an innovant mechanism of action, the inhibition of the mycobacterial ATP synthase;

• Bedaquiline has a delayed bactericidal activity on M. tuberculosis, and a sterilising efficacy which is synergistic with pyrazinamide;

• Bedaquiline has multiple clinically-relevant drug-drug interactions, in particular with antiretrovirals;

• The administration of bedaquiline increases the culture conversion rate and rate of favourable treatment outcome compared to placebo, according to Phase IIb trials;

• Bedaquiline has an overall good safety profile, although it can cause liver toxicity and prolongation of the QT interval;

• Multiple observational studies confirm these findings and some of them support the possibility of prescribing bedaquiline for extended durations and in combination with delamanid, if needed.

Delamanid

Structure and mechanism of action

Delamanid is the first compound of a class of anti-mycobacterials known as nitroimidazoles. It has been developed by Otsuka Pharmaceutical (Japan) through synthesis and screening of multiple compounds of this chemical class. 223 A second compound belonging to nitroimidazoles, pretomanid (formerly known as PA-824), is undergoing clinical development by the manufacturer. Since this drug is not currently available for routine use, it

will not be discussed in detail in this manuscript. Delamanid was known as OPC-67683

during drug development and is commercially available as Deltyba ® . His chemical 16).

composition is (R)-2-methyl-6-nitro-2-f4-[4-(4-trifluoromethoxyphenoxy)piperidin-1- yl]phenoxymethylg-2,3-dihydroimidazo[2,1-b]oxazole (Figure

Pharmacokinetics

Delamanid is a pro-drug, activated by the mycobacterial protein nitroreductase. Peak plasma levels are achieved 4-5 hours after administration. The drug disappears from the plasma with a half-life of 30-38 hours. The oral bioavailability of delamanid is good and improved by about 2.7-fold when administered with a standard meal, compared to fasting conditions. 222 Delamanid plasma concentration increases, although less than proportionally, with increasing doses. Delamanid has high binding properties with plasma proteins and a large volume of distribution in the tissues.

Interestingly, delamanid is primarily metabolised in plasma by albumin and only to a lesser extent by the liver by the CYP3A4 isoenzyme of the P450 cytochrome. 228 In addition, delamanid has no interaction with cytochrome P450 enzymes. 222 Albumin appears to degrade delamanid by binding to the electron-poor carbon at position 5 of nitrodihydroimidazooxazole and subsequent cleavage of the imidazooxazole moiety, leading to the formation of the main metabolite (M1). 229 Although the complete metabolic profile of delamanid has not been completely elucidated yet, there are at least eight delamanid metabolites that have been identified so far. None of them show anti-mycobacterial activity, but some appear to contribute to QT prolongation. Concentrations of the identified metabolites progressively increase to steady state after 6 to 10 weeks of treatment, as it has been shown by liquid chromatographic-tandem mass spectrometry. 230 The inhibition of mycolic acid production appears to be mainly exerted by a reactive intermediate metabolite,

formed between delamanid and its desnitro-imidazooxazole derivative. It has been demonstrated that neither delamanid nor its metabolites are transported by the main efflux ATP-binding cassette transporters, including P-glycoprotein. 231 Less than 5% of an oral dose of delamanid is found in the urine. Therefore, no dose adjustment is needed for patients with mild or moderate renal impairment. Similarly, no dose adjustment is considered necessary for patients with mild hepatic impairment. Delamanid is not recommended in patients with severe renal impairment or moderate to severe hepatic impairment. The finding that albumin plays an important role in the metabolism of delamanid has led the manufacturer and regulatory authorities to initially restrict the use of this drug to patients without hypoalbuminemia, mainly out of concern for increased risk of QT interval prolongation. Recent data seem to reduce the relevance of the influence of albumin on the tolerability of delamanid and of the QT prolongation risk associated with this drug in general. 232 Delamanid is a lipophilic compound and is therefore expected to have a satisfactory distribution in most of human tissues. This assumption has been confirmed by a study that investigated the distribution of a radioactive derivate of delamanide in mice, showing that delamanid and its metabolites penetrated in various tissues, including the central nervous system, eyeball, placenta, and foetus, as well as in maternal milk. 233 However, no evidence is available on humans on the penetration and activity of this drug on extrapulmonary forms of tuberculosis.

Drug-drug interactions

The limited role played by the liver in the metabolism of delamanid and the absence of effect of delamanid on cytochrome P450 enzymes suggest that this drug may have limited drug-drug interaction concerns. In addition, in vitro data showing little interaction between delamanid and transporters suggest that delamanid is unlikely to have clinically relevant interactions with drugs for which absorption and disposition are mediated by transporters. 231 A multiple-dose study was conducted in healthy subjects to assess the interaction between delamanid (and its metabolites) and antiretrovirals and first-line anti-tuberculosis drugs. Delamanid exposure was not affected by the coadministration of tenofovir or efavirenz; however, the coadministration of lopinavir/ritonavir resulted in approximately 25% higher delamanid AUC. Coadministration of delamanid with first-line drugs (rifampicin, isoniazid, pyrazinamide, and ethambutol) resulted in lower delamanid concentration (47% and 42% for AUC and Cmax, respectively), as well as decreased exposure of three primary metabolites (about 30 to 50% lower AUC). Delamanid did not affect the exposure of antiretrovirals and other tested anti-tuberculosis drugs. 234 As a result, it is generally recommended to try to avoid the co-administration of delamanid with strong inducers of the cytochrome P450, such as rifamycins, phenytoin, carbamazepine, and phenobarbital. Whenever avoiding the use of these drugs is not possible, the continuation of the anti-tuberculosis treatment should be assessed on a case-by-case basis.

The use of delamanid with inhibitors of the cytochrome P450, such as the protease inhibitors class among antiretrovirals, may lead to increase the exposure of delamanid; however, this is unlikely to have any clinical impact and the association is therefore not contraindicated (Tables 7 and8).

Pre-clinical studies: in vitro

Compared to bedaquiline, the amount of published evidence on the pre-clinical efficacy of delamanid for antimycobacterial treatment is limited. In the first description of delamanid, this compound was the most active in vitro among a large group of nitroimidazoles that were tested. 223 Subsequently, Matsumoto and co-workers showed that this drug inhibited the growth of drug-susceptible and drug-resistant M. tuberculosis strains at concentrations ranging from 0.006 to 0.012 mg/L. These MICs were lower than those achieved by pretomanid. In addition, delamanid was bactericidal also on intracellular mycobacteria in human macrophages and did not show any cross-resistance with other antimycobacterials. 222 In a Wayne dormancy model mimicking the environment of cellular granulomas, lipophilic drugs like nitroimidazoles were effective and significantly more active than hydrophilic drugs. 235 Consistently, delamanid was active on drug-tolerant M. tuberculosis strains in an in vitro model based on radiometric cultures, suggesting that this drug could have a sterilising activity. 236

Pre-clinical studies: animal model

The first experiments in the non-established TB infection mouse model confirmed the bactericidal activity of delamanid, which was comparable to the one of other first-line antituberculosis drugs. 222 These findings were replicated in the guinea pig model of chronic infection, where delamanid exerted bactericidal activity and showed to be effective also on hypoxic lesions in the infected lungs. 237 Multiple studies have assessed the sterilising activity of other nitroimidazoles, namely pretomanid and TBA-354, in the established TB infection mouse model. It is acceptable to assume that these findings could be at least partially applicable to delamanid. In a first study, several combinations of three and four drugs among bedaquiline, pretomanid, sutezolid, and clofazimine, were tested. Combinations including pretomanid led to a faster sterilization than a first-line regimen of rifampin, pyrazinamide, and isoniazid. 180 A second study suggested that the addition of a nitroimidazole between pretomanid and TBA-354 significantly improved the sterilizing activities of bedaquiline and sutezolid, with or without pyrazinamide.

It also suggested that TBA-354 activity might be superior to the one of pretomanid. 182 Finally, a third study confirmed that the combination of bedaquiline, pretomanid, moxifloxacin, and pyrazinamide, has a promising sterilizing activity that allowed to render all tested mice relapse-free after only two months of treatment. 183 dosages appeared after two days of treatment. Delamanid EBA was maximal for the 200 mg and 300 mg daily arms and increased with a less than dosage-proportional relationship, probably linked to an upper threshold in dose absorption. All delamanid arms had a significantly lower EBA than the standard anti-tuberculosis treatment arm (Figure 17). 238 In two other Phase IIa studies, pretomanid was tested as part of multidrug combinations. These results might be applicable to delamanid as well, in light of the many similarities between the two compounds. In one study, the 14-day EBA was assessed in six groups receiving 1) bedaquiline, 2) bedaquiline-pyrazinamide, 3) pretomanid-pyrazinamide, 4) bedaquilinepretomanid, 5) pretomanid-moxifloxacin-pyrazinamide, and 6) standard antituberculosis treatment. The pretomanid-moxifloxacin-pyrazinamide arm showed the highest EBA, which was comparable to that of the pretomanid-pyrazinamide and standard antituberculosis arms, and superior to the bedaquiline-containing arms. 193 In the second study, the 14-days EBA was compared across seven treatment arms: 1) bedaquiline-pyrazinamide-clofazimine; 2) bedaquiline-pretomanid-pyrazinamide; 3) bedaquiline-pretomanid-pyrazinamide-clofazimine; At week eight of treatment, 45.4% of patients in the delamanid 100 mg arm had sputum culture conversion in liquid medium, compared to 41.9% in the delamanid 200 mg arm, and 29.6% in the placebo arm. Both delamanid 100 mg and delamanid 200 mg showed a significantly superior 8-week efficacy compared to placebo (p=0.008 and p=0.04, respectively) (Figure 20). These differences were confirmed on the analyses of sputum culture conversion rates on solid medium and of time to sputum culture conversion. 67 In the analysis, participants who received delamanid for at least six and up to eight months were compared to participants who did not receive delamanid or who received it for up to two months: favourable outcomes were significantly higher (74.5%) in the former than in the latter (55%) group. In addition, mortality was lower in the longer delamanid treatment group (1%) than in the shorter delamanid treatment group (8%). 240 The association between longer delamanid treatment and increased long-term survival was confirmed by a subsequent analysis. 241 Trial 213 is a Phase III trial comparing delamanid plus optimized background regimen to placebo plus optimized background regimen in pulmonary MDR-TB patients. Notably, delamanid was given at 100 mg twice daily for the first two months, and subsequently at 200 mg once daily for the rest of the treatment, for a total of no more than 24 weeks. The primary outcome was time to sputum culture conversion, and secondary outcomes were culture conversion rates at 2 and 6 months, and treatment outcome. Unpublished Phase III trial data have been recently presented at a conference. 232 Although not well detailed, the main results of the study are therefore publicly available. Overall, 511 participants were randomized, 341 in the delamanid arm and 170 in the placebo arm: 327 participants were finally retained in the modified intention-to-treat population, the main reason for exclusion being culture-negative at treatment start. The study population also included fluoroquinolone-resistant MDR-TB and XDR-TB cases, which were more frequent in the delamanid than in the placebo arm (11.5% vs 6.0%, respectively). The primary outcome, time to sputum culture conversion, showed a modest improvement in the delamanid arm compared to the placebo arm, although the median difference (six days) was not statistically significant. The difference was greater (13 days) and statistically significant in two sensitivity analyses (p=0.028, 0.005). Sputum culture conversion rates at two and six months were higher in the delamanid than in the placebo arm,

although not significantly so. The study was not powered for long-term outcomes and these were very similar in the experimental (81.4%) and placebo (81.2%) arms. In an exploratory analysis, the delamanid arm showed less amplification of drug resistance during treatment than the placebo arm, although the overall difference was not statistically significant. Table 9 summairzes the efficacy outcomes of the delamanid trials. As elucidated in Table 10, the vast majority of published papers reporting the efficacy results of observational studies of MDR-TB patients treated with delamanid have been published after 2016. This is consequent to the slow introduction of this drug in both routine and compassionate use programs worldwide. The growth in available evidence in the last twothree years is remarkable, although the overall amount of research is still lagging when compared to bedaquiline. Studies reporting treatment efficacy in special populations (i.e. pediatric) or "off-label" use of delamanid will be described into detail later.

Overall, 15 papers were identified at the time of this review. Six of them were case reports or case series describing five patients or less, seven were retrospective cohort studies reporting mainly or solely interim treatment efficacy results, and two were retrospective cohort studies reporting final treatment outcome.

In the first retrospective cohort describing end-of-treatment outcome, nine out of 11 (81.8%) MDR-TB patients receiving delamanid in Hong Kong were cured, while one failed treatment, and another was still undergoing treatment at the moment of data censoring. The patient who failed treatment likely developed resistance to both linezolid and delamanid, probably due to a weak background regimen, and eventually died. Overall, eight patients received more than 24 weeks of delamanid. 242 In the second study, a retrospective cohort including 19 MDR-TB patients from Latvia, 16 patients (84.2%) were classified as cured, three patients (15.8%) were lost to follow-up, and there were no deaths or treatment failures.

Of note, 10 patients received prolonged (>24 weeks) treatment duration with delamanid and two patients received both bedaquiline and delamanid. 243

Among the seven cohort studies analysing interim efficacy outcomes of delamanidcontaining MDR-TB treatment regimens, three found sputum culture conversion rates at six months of treatment ranging between 67.6% and 94.4% of patients having a positive sputum culture at delamanid treatment start, 200,245,246 two found that 80% and 81.2% of patients had negative sputum cultures at 24 weeks of treatment, 244,248 one found that culture conversion rate was 70.9% in a cohort including patients receiving bedaquiline, delamanid, and both, 200 and one found that, out of 28 patients treated with the combination of bedaquiline and delamanid, 74% achieved culture conversion at six months of treatment. 201

Among the six case reports and case series, one described the access of a paediatric MDR-TB case to delamanid through compassionate use, but without any detail on treatment efficacy. 249 The others, three describing treatment with the bedaquiline/delamanid combination 206,209,213 and two describing an MDR-TB case who failed treatment and acquired sequentially resistance to both bedaquiline and delamanid, 218,219 were described before in the bedaquiline section.

Safety: clinical trials

In the three Phase IIa EBA studies including nitroimidazoles, the tolerability of the arms containing delamanid or pretomanid was overall good, although it should be noted that the follow-up period was of two weeks only. In the first study, delamanid was well tolerated without serious adverse events at all dosages. 238 In the second study, adverse events were commonly reported but were mostly mild; overall, seven patients were withdrawn from the study for toxicity, including five cases with alanine aminotransferases over three times the upper limit of normal, one patient receiving pretomanid-moxifloxacin-pyrazinamide who had a >60 ms QT and was withdrawn from the study, and one patient with neurocysticercosis. 193 In the third study, adverse events were also common (62% of participants), with seven presenting severe (grade three or four) adverse events. Among those, a participant receiving bedaquiline-pretomanid-pyrazinamide experienced a grade three rise in transaminase values, and two others receiving bedaquiline-pretomanid-pyrazinamide and bedaquiline-pretomanidclofazimine experienced a grade four rise in transaminase values. Overall, a >60 ms QTcF increase from baseline was reported for five participants, four of which were in the bedaquiline-pretomanid-clofazimine arm. 194 In the Phase IIb study assessing the eight-week bactericidal activity of different treatment combinations including pretomanid, most patients (88%) had at least one adverse event, nine (4%) patients had a serious adverse event, and one died. The death, which occurred 6 weeks after the discontinuation of the study treatment, was not assessed by the investigator as treatment-related. No QTcF of more than 500 ms were reported, although >60 ms QTcF increases from baseline were reported for two (3%) patients in the moxifloxacinpretomanid 100 mg-pyrazinamide arm and four (7%) patients in the moxifloxacin-pretomanid 200 mg-pyrazinamide arm among the drug-susceptible patients, and for two patients receiving moxifloxacin-pretomanid 200 mg-pyrazinamide (8%) among drug-resistant patients. 239 In the pivotal Phase IIb study comparing delamanid at 100 mg and 200 mg twice daily for eight weeks to placebo (Trial 204), the safety population was composed by 481 patients.

Overall, 93.3% of participants experienced at least an adverse event, 10.4% experienced at least a serious adverse event, and 2.9% discontinued the study treatment because of adverse events. All these instances were evenly distributed across the three arms, with a slightly higher frequency in the delamanid 200 mg arm. The rate of QTcF prolongation was higher in the 200 mg delamanid arm (13.1%) than in the 100 mg delamanid arm (9.9%), and both rates were higher than that in the placebo arm (3.8%). No episodes of a prolonged QTcF interval were associated with clinical manifestations such as syncope or arrhythmias. [START_REF] Gler | Delamanid for multidrug-resistant pulmonary tuberculosis[END_REF] Safety data from Trial 208 and Observational Study 116 are unfortunately unavailable.

However, the investigators performed an additional analysis including all available data from the three studied composing the clinical development program of delamanid to assess the impact on the QT interval. Overall, delamanid QT-prolonging action peaked at 8 weeks of treatment, and in no case was associated with clinical manifestations. The concomitant use of levofloxacin or clofazimine did not have a significant impact. 250

In the Phase three Trial 213, the safety findings in the delamanid arm were reassuring.

There was no significant difference between delamanid and placebo arms in the incidence of total adverse events, serious adverse events, in the number of adverse events that led to treatment discontinuation, and in the cases of hepatotoxicity. In the delamanid arm there was a greater rate of QT interval prolongation than in the placebo. However, the difference in QT prolongation between delamanid arm and placebo arm at eight weeks was lower in Trial 213

(5.3 ms) than in Trial 204 (12.1 ms), and decreased at week 24 (2.5 ms) 232 . The safety results

of the delamanid trials are summarized in Table 11.

Safety: observational studies

In the retrospective cohort from Hong Kong, the safety of the treatment is not well described, although generally defined as satisfactory. No treatment discontinuation because of QT prolongation were reported. As described above, one patient died after having failed treatment and likely developed resistance to both delamanid and linezolid. 242 In the retrospective cohort from Latvia, the rate of adverse events is not described. Overall, no clinically relevant adverse event was attributed to delamanid, no case of hepatotoxicity occurred, and no QTcF>500 ms was reported. 243 In a multinational cohort including 53 MDR-TB patients treated with delamanid for 24 weeks, a total of 31 serious adverse events were reported in 14 patients (26.4% of total). The most common among them were hepatotoxicity, electrolyte imbalance, and QT prolongation.

There were seven reported deaths, and two of them were deemed by the investigator as possibly related to anti-tuberculosis drugs: a respiratory failure related to end-stage hepatitis and a sudden death of unknown cause. Permanent treatment discontinuation occurred in one patient with hepatitis C and liver cirrhosis following hepatotoxicity. 245 In another multinational review including interim results of 78 MDR-TB patients treated with delamanid under compassionate use, QT>500 ms was reported in three (3.8%) patients: delamanid was permanently discontinued in two cases and temporarily before being reintroduced in one case.

Overall, eight patients died prior to the completion of the 24-week treatment course: none of these deaths was considered by the treating clinician as likely caused by delamanid. 244 In a cohort of 103 MDR-TB patients treated with delamanid in South Africa, there were 67 serious adverse events reported in 29 (28%) patients: of these, 22 (33%) were attributed to delamanid, and the most common were QT prolongation and vomiting. There were three hepatic serious adverse events. Overall, QTcF>500 ms occurred in two patients, in one of whom delamanid was permanently withdrawn. No cardiac arrhythmias nor clinically-relevant cardiac adverse events were reported. 246 In a cohort from South Korea, 25 out of 32 patients (78.1%)

receiving delamanid experienced at least one adverse event. No serious adverse event or death were observed. Four patients transiently discontinued delamanid, two of whom following QTcF prolongation >500 ms. 247 In a multinational cohort of adolescents and children treated with delamanid in compassionate use programs, the overall tolerability was good, except for one patient who experienced severe vomiting, renal impairment, electrolyte disturbances, and QTcF>500 ms requiring temporary delamanid discontinuation. After the correction of the electrolyte imbalance, the patient was able to complete delamanid treatment. 248 In a retrospective cohort from South Korea, 61 patients received either bedaquiline delamanid, or both, as part of MDR-TB regimens. The overall safety results were described before in the bedaquiline section. 200 Results from the multinational cohort describing interim results of the bedaquiline/delamanid combination were also described before. 201 The case report describing compassionate use of delamanid did not include any safety information. 249 The safety finding of the three studies describing treatment with the bedaquiline/delamanid combination 206,209,213 and of the two studies describing the MDR-TB case with treatment failure and resistance acquisition, 218,219 were described before in the bedaquiline section.

In summary:

• Delamanid, together with pretomanid, belongs to a drug class with an innovant mechanism of action, the inhibition of the synthesis of the mycobacterial cell wall;

• Delamanid has a weak bactericidal activity on M. tuberculosis, while sterilising activity has only been demonstrated for pretomanid so far;

• Delamanid has few clinically-relevant drug-drug interactions;

• Delamanid administration leads to faster culture conversion, compared to placebo, in Phase II and III clinical trials; improved treatment outcomes where demonstrated in the follow-up of a Phase IIb trial, but not confirmed by a subsequent Phase III clinical trial;

• Delamanid has an excellent safety profile, with only mild increase of the QT interval and of the rate of gastrointestinal adverse events;

• Some observational studies confirm these findings, and a few support the possibility of prescribing delamanid for extended durations and in combination with bedaquiline, if needed.

"Off-label" use and use in special populations

Current indications for bedaquiline and delamanid

Following FDA approval, bedaquiline is currently indicated "…as part of combination therapy in adults (≥ 18 years) with pulmonary MDR-TB". 220 The dosage is, according to the drug package insert, "400 mg once daily for two weeks followed by 200 mg three times per week for 22 weeks with food". The indication for delamanid, as formulated by the European Medicines Agency (EMA), is "…use as part of an appropriate combination regimen for pulmonary MDR-TB in adult patients…", at a dosage of "100 mg twice daily for 24 weeks".

These indications are based on the evidence provided by the pivotal trials that supported the efficacy and safety of these two drugs, namely study C208-2 for bedaquiline and Trial 204 for delamanid. Although there is widespread consensus that the new drugs bedaquiline and delamanid should be reserved (at least for now) to the treatment of active drug-resistant tuberculosis, the indications for their use are quite narrow, and have only been Taking into account these circumstances and the special case of bedaquiline and delamanid, the WHO has issued a specific document to give guidance on the off-label use with these two drugs. According to this document, in the absence of high-quality evidence, "off-label use" is justified when:

• the target condition is serious and there is other evidence to support potential benefit (particularly if the expected benefits outweigh potential risk);

• standard therapy is judged to be inadequate to achieve the desired outcome;

• patients have been duly informed about the potential benefits and risks of "offlabel use" and have consented (consent in writing may be necessary in some settings);

• active and adequate monitoring of medicine safety is in place, including mechanisms to rapidly identify and manage adverse events. 251 In this chapter, we will review the available evidence supporting the most common categories of "off-label" use or of use with expanded indications for bedaquiline and delamanid.

Use of bedaquiline and delamanid in children

No children were included in the above-mentioned pivotal trials that led to the approval of bedaquiline and delamanid. Consequently, both of these drugs were initially indicated only for adults.

There is currently very little evidence for the use of bedaquiline in children, although it is commonly acknowledged that, in the absence of therapeutic alternatives, bedaquiline may promising efficacy and safety results. 248 In addition, paediatric patients were included in many other delamanid-treated cohorts and case reports. 243,245,246,249

Use of bedaquiline and delamanid in extrapulmonary tuberculosis

Clinical trials testing anti-tuberculosis drugs are typically restricted to patients with pulmonary tuberculosis, and in particular to those with a positive sputum culture at baseline. This is a pragmatic choice, in light of the fact that treatment outcomes are mostly based on microbiological outcomes and therefore require the availability of regular specimens (i.e.

sputum samples) during follow-up. This has been the case for all bedaquiline and delamanid trials, leading to the approval of these drugs for pulmonary tuberculosis forms. However, many clinicians have used the drugs off-label also for extrapulmonary cases, whenever needed. In the interim guidance for both bedaquiline and delamanid, the WHO supports this pragmatic approach stating that "while patients with exclusive extrapulmonary disease were not included in the … trial, the use of the drug in extrapulmonary tuberculosis patients may be considered, extrapolating from the data in patients with pulmonary tuberculosis." 253,254 It should be noted, however, that there is very limited knowledge on the penetration of the new anti-tuberculosis drugs in specific tissues such as bone, cerebro-spinal fluid, brain, pleural liquid, and others.

Use of bedaquiline and delamanid for extended duration

All Phase IIb and III trials testing the new anti-tuberculosis drugs have assessed two or six-month courses of bedaquiline/delamanid. These durations of the treatment courses have been chosen according to the primary outcome of the studies, which in these studies was a surrogate outcomeeither the rate of sputum culture conversion at two or six months, or the time to sputum culture conversion. Culture conversion rates, in particular at the six-month endpoint, have been shown to be acceptable markers for end of treatment outcome, although lacking specificity. [255][256][257] The main advantage of using surrogate markers as the primary outcome of clinical trials is, pragmatically, that they allow recruiting a smaller sample size, resulting in earlier results and reducing the overall costs. The overall evidence originating from such trials is, inevitably, of lower quality than those originating from trials whose primary outcome is the end-of-treatment outcome. This has sparked long debates in the scientific community at the moment of the provisional approval of bedaquiline by the FDA. 258 It is also controversial that a Phase III trial, such as delamanid Trial 213, would adopt a surrogate outcome measure (time to sputum culture conversion) as its primary outcome. The result of this development policy by the manufacturers is the recommendation by regulatory authorities and the WHO to use bedaquiline and delamanid for a standardized duration of 24 weeks. 254,259 The choice of restricting the duration to 24 weeks is usually justified by safety worries, in particular in relationship with the long terminal half-life of bedaquiline. In a multidrug regimen such as the one for M/XDR-TB, however, this strategy contradicts the principles of antimicrobial polychemotherapy, where drugs are usually associated for the full duration of treatment to prevent the emergence of drug resistance. In a cohort of patients treated with bedaquiline on compassionate use with the support of Médecins Sans Frontières in Armenia and Georgia, and presented recently, almost 20% of 53 MDR-TB patients who had achieved sputum culture conversion experienced reversion to positive cultures after interrupting bedaquiline treatment at 24 weeks. 260 Very little evidence is available so far on off-label prolonged treatment courses of bedaquiline/delamanid: only a case report for bedaquiline, 214 and some patients in small cohorts for delamanid. 242,243 No safety signals were detected in these patients.

Use of bedaquiline and delamanid in pregnant women

TB and MDR-TB are common among women during their child-bearing years. They require treatment, which often cannot be safely deferred until after pregnancy. Information on regimens optimized for safety and efficacy in this population, however, is almost nonexistent, as illustrated by the presence in Category C of nearly all drugs used routinely for the treatment of MDR-TB, including in pregnant women. For bedaquiline, reproductive toxicity studies have not shown teratogenicity but data in humans is lacking. Only a single case report is currently available of an MDR-TB patient who received bedaquiline since week 36 of pregnancy: the mother was cured, and the child did not show any growth or other clinical disorders after two years of follow-up. 207 Delamanid was teratogenic in reproductive toxicity studies, although this has not been confirmed in humans so far.

Therefore, in case a new anti-tuberculosis drug is needed to design an effective regimen in a pregnant woman, it is generally recommended to use bedaquiline and monitor closely the evolution of the pregnancy and of the newborn.

Use of bedaquiline and delamanid in combination

The safety profile of bedaquiline combined with delamanid has not been studied and the two drugs were not given in combination in any of the manufacturer-sponsored clinical trials. Although not formally opposing the combination use of bedaquiline and delamanid, the WHO states that "concomitant use should be reserved for regimens of last resort in patients with extensive patterns of drug resistance, drug intolerance or serious adverse effects". 251 In the last version of the Companion Handbook to the MDR-TB guidelines, the suggested approach is observing a washout period of 5 days when switching from delamanid to bedaquiline and of six months when switching from bedaquiline to delamanid. 261 However, the sequential use of any new antibiotic raises the risk of selecting drug resistance, in particular in presence of a period of washout between one drug and the other. This risk is exemplified by a published clinical case of an MDR-TB patient receiving sequentially bedaquiline (which was added late to an ineffective regimen) for 24 weeks and then delamanid, failing twice and developing drug resistance to both drugs. [217][218][219] Until the end to 2016, only two case reports of XDR-TB patients treated with the combination of bedaquiline and delamanid were published. In one case, a pulmonary XDR-TB patient, the association was well tolerated and no QT prolongation occurred. 209 In the second case, QT interval prolongation was observed during XDR-TB treatment with a drug combination including clofazimine, bedaquiline and delamanid; following the QT increase, bedaquiline was interrupted and re-introduced after five days. No QTcF>500 ms was recorded, nor any cardiac arrhythmia occurred. 213 In 2017, a case series of five MDR-TB patients treated with concomitant bedaquiline/delamanid treatment was published: one patient was cured, three were still on treatment at data censoring, and one failed treatment by developing additional drug resistance to linezolid and ethionamide and eventually died of respiratory failure. Treatment tolerability was overall good, with no cardiac events except for QT prolongation >500 ms in one patient. 206 Recently, a multinational cohort of 28 adult MDR-TB patients treated with the support of Médecins Sans Frontières and receiving the bedaquiline-delamanid combination has been published. Only interim outcomes were presented, with a culture conversion rate of 74% at six months of treatment. Remarkably, treatment tolerability was good, with no case of QTcF>500 ms. Four patients had QTcF increase of more than 60 ms from baseline but none permanently discontinued the drugs.

In conclusion, to date, no cardiac events have been reported among patients receiving either bedaquiline, delamanid, or both. ACTG 5343, the National Institute of Allergy and Infectious Diseases (NIAID)-sponsored combination trial of bedaquiline and delamanid, will provide the first evidence from a well-conducted trial of the safety and efficacy of delamanid and bedaquiline in combination. That trial, however, has encountered significant delays and has only recently started enrolling patients.

Development of drug resistance to bedaquiline and delamanid

As frequently happened in the history of chemotherapy for infectious diseases, drug resistance has been following shortly after the introduction of the new anti-tuberculosis drugs bedaquiline and delamanid. Drug resistance mechanisms, initially studied in vitro and in the animal model, have been progressively elucidated by analysing clinical isolates and relating laboratory testing and clinical outcome. Up to date, however, the optimal methods for performing and interpreting phenotypic and genotypic drug susceptibility testing for bedaquiline and delamanid are not established yet. 262 Standard procedures to perform phenotypic drug susceptibility testing for bedaquiline and delamanid have been recently

proposed for MGIT960 liquid culture medium [263][264][265] and for solid media, [266][267][268] although not validated yet. In a retrospective study, isolates from trials C208 and C209 were retrieved and gene rv0678 was systematically sequenced: surprisingly, mutations were identified in 6.3% of MDR-TB baseline isolates and even in 0.7% of drug-susceptible isolates. Bedaquiline MICs for isolates harbouring rv0678 mutations were very variable, comprising high, normal, and low values. The authors concluded that these results questioned the utility of genotypic testing to detect clinically meaningful resistance to bedaquiline. 275 In another study, five clofazimineresistant isolates, four of which were also resistant to bedaquiline, were isolated from pre-XDR and XDR-TB patients with no documented prior exposure to clofazimine or bedaquiline. All four isolates exhibiting cross-resistance harboured a mutation in rv0678, while the other harboured a mutation in the rv1979c gene. This led the authors to postulate that rv0678 mutations might be selected by treatment with other anti-tuberculosis drugs. 276 Similarly, out of 90 XDR-TB clinical isolates analysed in a centre in China, five were resistant to clofazimine, all harboured a mutation in rv0678, and all had increased bedaquiline MICs. 277 In a study performed in Russia, 24 clinical isolates with increased bedaquiline MICs were analysed, showing mutations in rv0678 (by far the most frequent), atpE, atpC, pepQ, and rv1979c. 278 Recently, a large study from South Africa was published, presenting the results of phenotypic testing for bedaquiline and whole genome sequencing for 387 isolates from bedaquiline-naïve patients and 14 isolates from bedaquiline-exposed patients. Among isolates from bedaquiline-naïve patients, no atpE mutations were detected while 3 (0.8%), 4

Drug resistance to bedaquiline

(1.1%), and 98 (25.9%) mutations were observed in the rv0678, pepQ, and rv1979c genes, respectively. However, only two isolates with rv0678 mutation and 1% of the isolates with rv1979c mutation had increased bedaquiline MICs. Interestingly, among the eight isolates from bedaquiline-exposed patients who had slow treatment response and potential treatment failure (sputum culture positive at six months), all had bedaquiline MICs at or above the intermediate category and six harboured a mutation in the rv0678 gene. Among clofazimineresistant isolates, 30% were also resistant to bedaquiline, while all bedaquiline-resistant strains were also resistant to clofazimine, suggesting that the most common resistance mechanism for clofazimine does not involve cross-resistance with bedaquiline. 279 Interestingly, advanced studies on the metabolic and transcriptomic modifications adopted by M. tuberculosis while exposed to bedaquiline support the efficacy of combined chemotherapy including diarylquinolines and other agents. In one study, it was shown that bedaquiline-mediated inhibition of the mycobacterial electron transport chain leads M.

tuberculosis to reroute by switching between terminal oxidases and increasing total respiration to maintain ATP levels. This increase in activity potentiates clofazimine's production of reactive oxygen species, causing rapid killing in vitro and in a macrophage model, and suggesting a synergic action between these two drugs. 280 A second study has observed how exposure to bedaquiline induces mycobacteria to adopt a tolerant state through the activation of transcription factors rv0324 and rv0880. Disruption of this reactive network significantly increased bedaquiline killing. Elegantly, the authors show that a nitroimidazole like pretomanid inhibits the rv0880 regulon, thus potentiating bedaquiline activity. 281

Drug resistance to delamanid

The frequency of spontaneous resistance to delamanid was initially estimated between 10 -5 and 10 -6 , similar to the one associated with isoniazid or pretomanid. In the first in vitro studies, delamanid resistance was associated with mutations in the ddn and/or fgd1 genes. 282 When the first clinical case of acquired drug resistance to delamanid was described, resistance to delamanid was associated with mutations in the fbiA and fgd1 genes. 218 After deep sequencing of the clinical isolates, the D49Y mutation in fbiA was identified as the resistanceconferring one. 219 FbiA, like the other genes mentioned above, is involved in the biosynthetic pathway of the flavin cofactor F420, which metabolizes and activates delamanid and pretomanid (which are prodrugs). In a study of clinical XDR-TB isolates, five delamanidresistant isolates were identified, and they all harboured a mutation in codon 318 of the fbiC gene. 277 A recent study has confirmed that ddn, fgd1, fbiA, fbiB, and fbiC genes are all involved in the development of drug resistance to delamanid and pretomanid. 283

Approvals and recommendations for the use of bedaquiline and delamanid

Bedaquiline has received conditional approval from the FDA in 2012. The After the approval of bedaquiline and delamanid for MDR-TB treatment by the FDA and the EMA, respectively, the WHO has issued interim recommendations to guide the introduction in the clinical practice of bedaquiline in 2013 and of delamanid in 2014. 254,259 The review of the available data led to conclude in both documents that the evidence was of insufficient quality to make any strong recommendation on the use of these drugs. In both cases, the WHO underlined the commitment to update the provisional guidelines as soon as sufficient evidence would become available. Overall, there was specific concern with regard to the safety bedaquiline, in light of the mortality excess in the experimental arm of trial C208 stage 2.

The conditional recommendation, common to both documents, was to use bedaquiline or delamanid in adult patients with pulmonary MDR-TB:

• when an effective treatment regimen containing four second-line drugs in addition to pyrazinamide according to WHO recommendations cannot be designed;

• when there is documented evidence of resistance to any fluoroquinolone in addition to multidrug resistance.

In addition, delamanid was recommended also for patients with resistance to secondline injectables (even in absence of fluoroquinolone resistance) and for those with higher risk for poor outcomes (e.g. drug intolerance or contraindication, extensive or advanced disease).

In both cases, a series of conditions would have to be fulfilled:

• Treatment administered under closely monitored conditions;

• Proper patient inclusion (according to criteria above);

• Patient informed consent obtained;

• Adherence to principles of designing a WHO-recommended MDR-TB regimen;

• Pharmacovigilance and proper management of adverse drug reactions and prevention of drug-drug interactions.

These drug-specific recommendations were revised once for both drugs. In 2016, on the grounds of new pharmacokinetic data provided by the manufacturer, a new document was issued to support the use of delamanid in children and adolescents between 6 and 17 years of age. Recommendations on the adapted posology were also provided. This document did not change the eligibility criteria and implementation requirements as listed above. 253 In 2017, an update of the recommendations for the use of bedaquiline was provided based on evidence from cohorts of MDR-TB patients who received bedaquiline in Armenia, France, Georgia, South Africa, and in multiple countries as part of the C209 trial. Overall, the indications and implementation requirements were not modified: however, the guideline review committee noted the availability of preliminary evidence on the safety of the use of bedaquiline in adolescents and for extended duration (>24 weeks) and downgraded the anticipated undesirable effects of bedaquiline from "large" to "moderate". 252 Source: WHO 2018 284 In 2016, the overall RR/MDR-TB recommendations were revised by the WHO. This revision included the change in the grouping of the drugs: bedaquiline and delamanid, previously in Group 5 (drugs with unclear efficacy, not recommended for routine use), were moved to group D2, immediately after the core second-line drugs, although the abovementioned indications were not modified. 285 The recent issue of a rapid communication on the key changes in the treatment of RR/MDR-TB has completely revolutionized the grouping of the drugs. Based on the results of a meta-analysis including individual patient data from over 12000 MDR-TB patients that showed reduced mortality and higher likelihood of treatment success in patients receiving bedaquiline, bedaquiline was upgraded to Group A, which includes the medicines to be prioritised. Therefore, bedaquiline will likely be one of the core drugs that will form the main nucleus of any MDR-TB treatment, except for patients with specific contraindications. Delamanid is now classified in Group C, which is composed by medicines to be included to complete the regimens and when agents from Groups A and B cannot be used (Table 12). Individual patient data from the Phase III trial 213 were however not analysed in the preparation of the 2018 update: the WHO will re-assess the role of delamanid once these data will become available. 284 Provided by the Working Group on New TB Drugs (www.newtbdrugs.org).

Indeed, the current pipeline of new anti-tuberculosis drugs in development (Figure 23)

may seem rich at a first glance. However, when investigated in detail, the overall outlook is much less promising. Apart from bedaquiline and delamanid, the only other compound that is undergoing Phase III of clinical development is pretomanid. However, pretomanid is a nitroimidazole like delamanid and is very likely to share the same resistance mechanism. The two nitroimidazoles will be therefore alternative but not be used in combination. Among compounds in Phase II of clinical development, two (delpazolid and sutezolid) are oxazolidinones like the re-purposed drug linezolid. These drugs have the same resistance mechanism as linezolid and are mainly being studied to identify a less toxic alternative to linezolid. SQ109 is a novel 1,2-ethylene diamine small molecule with a distinct mechanism of action. However, after having shown promising activity in vitro and in animal studies, 145,286 a 14-day EBA study reported no activity of SQ109 alone or in combination with rifampicin. 287 In addition, in a recent multi-arm multi-stage study testing different treatment regimens including high-dose rifampicin, ethambutol, moxifloxacin, and SQ109, the arms containing SQ109 were dropped for insufficient efficacy. 288 The clinical development of this drug is therefore likely to be discontinued by the company. Macozinone is a piperazinobenzothiazinone derivative that inhibits DprE1, an enzyme that is essential for the 13 for fluoroquinolone-susceptible MDR-TB and in Table 14 for fluoroquinolone-resistant XDR-TB. For fluoroquinolone-susceptible MDR-TB, there are four pivotal Phase III clinical trials that are currently ongoing, and that have the potential to revolutionize the currently-recommended treatment. These trials are testing different combinations including at least one and up to two new drugs among bedaquiline, delamanid, and pretomanid; the total duration of tested treatment is six to nine months, and the control is represented by WHO-recommended standard-of-care regimen. Two trials, both sponsored by Médecins Sans Frontières, have an innovant design that aims at increasing the scientific yield while minimizing the sample size. 289,290 The first, TB-PRACTECAL (clinicaltrials.gov: NCT02589782), is a multi-arm, multi-stage trial that tests three experimental regimens: one or two of the experimental arm will be dropped based on the culture conversion rate at 8 weeks, while the other one/two arm/s will be continued until the end of the follow-up. The second, endTB (clinicaltrials.gov: NCT02754765), is a six-arm Bayesian response-adaptive trial, a design that is prevalently used in oncology trials: in Bayesian randomization, the patients' likelihood of being randomized to each experimental arm is linked to the performance of the arm at interim outcomes (week 8 and week 39) and re-assessed every month of recruitment. 291,292 The number of studies recruiting fluoroquinolone-resistant MDR-TB patients is smaller. Among them, only two are controlled trials, and therefore having reasonable ambitions of conducting to a change in treatment recommendations. One is the aforementioned trial TB-PRACTECAL, which is however not powered to draw any specific conclusion on fluoroquinolone-resistant MDR-TB population. The other is endTB-Q, a trial testing a four-drug regimen containing bedaquiline, delamanid, linezolid, and clofazimine, given for six and nine months, respectively, in two experimental arms.

Overall, it is crucial that the rapid implementation of new drugs and regimens, as soon as they will be supported by sufficient evidence, will be provided together with support from national programs and international agencies to promote training programs and antimicrobial resistance awareness campaigns. The optimal management of each individual patient, together with systematic surveillance of drug resistance and quick adaptation to epidemiological changes in the population, is the key to avoid the quick spread of drug resistance which has led to the failure of many efforts to control tuberculosis in the past. 293,294 (97%) cases after 6 months of bedaquiline-containing treatment; median time to sputum culture conversion and time to sputum smear conversion were 85 (range, 8-235 days) and 103 days (range, 12-252 days), respectively. In multivariable logistic regression, presence of a fluoroquinolone in the treatment regimen (p=0.01), absence of lung cavities (p<0.001), and absence of hepatitis C virus infection (p=0.001) were significantly associated with faster sputum culture conversion. Overall, 7 patients (20%) experienced a ≥60 ms increase in QTcB interval, leading to bedaquiline discontinuation in two (6%) cases; however, no cardiac arrhythmias were recorded. Severe liver enzyme elevation occurred in two patients (6%). One patient died: the death was reported by the clinicians as unrelated to TB or to anti-TB treatment.

The results of the study were overall promising. Interim efficacy results showed that the use of individualised treatment regimens including bedaquiline together with other repurposed drugs like linezolid may achieve sputum culture conversion even in MDR-TB patients with advanced pulmonary TB and complicated resistance patterns. The safety profile of the treatment was reassuring, with no major cardiac events being reported, although adverse events were frequent. However, the short follow-up time called for surveillance until the end of the treatment for comprehensive assessment of bedaquiline safety and efficacy.

drugs. Overall, 36 patients (80%) had a favourable treatment outcome, five were lost to follow-up, three died, and one failed treatment and acquired bedaquiline resistance. No relapses were reported. Severe and serious adverse events were recorded in 60% and 18% of patients, respectively. QTcF values >500 ms were recorded in 11% of patients, but neither arrhythmias nor symptomatic cardiac side-effects occurred. Bedaquiline was discontinued in three patients following QTcF prolongation. No significant differences were recorded between patients receiving standard and prolonged bedaquiline duration in the incidence of total, severe and serious adverse events, including liver enzyme elevation. No statistical difference was found between the two groups in the rate of >60 ms QTcF prolongation and of QTcF >500 ms values, nor in the maximum QTcF increase recorded during treatment.

Patients in the prolonged treatment group had slower time to culture conversion (91 versus 71 days; p=0.021); however, favourable treatment outcome rates at the end of treatment and during post-treatment follow-up were comparable between the two groups.

The results of this study provide evidence supporting the use of bedaquiline as part of treatment regimens for M/XDR-TB. Although total rates of adverse events were high, the cardiac safety profile was satisfactory. Remarkably, no increase in toxicity was reported in the group of patients receiving prolonged bedaquiline treatment. In addition, even if the group of patients receiving prolonged bedaquiline presented a number of characteristics associated with worse outcomes, and achieved culture conversion later during treatment, the rate of favourable outcomes was comparable between standard and prolonged bedaquiline groups.

These findings provide new evidence to support the use of bedaquiline in M/XDR-TB, as a majority of difficult-to-treat patients had favourable outcome without major adverse events. In addition, this was among the first, and at the time of the literature search the largest, study to show the efficacy and safety bedaquiline treatment prolonged beyond six months in selected cases when no alternatives are available.

VII. Article 3

Is bedaquiline as effective as fluoroquinolones in the treatment of multidrug-resistant tuberculosis? sequentially. Compared with controls, cases were more likely to be male, born in Eastern Europe, previously treated for TB, to have bilateral pulmonary involvement, and to harbour strains with more additional resistance; there was no difference in the presence of lung cavities and sputum smear status at treatment start. Three patients were HIV positive: they were all among the controls. Cases received less frequently ethambutol and ethionamide, but more frequently clofazimine and the combination of a carbapenem and clavulanic acid. The three-month culture conversion rate was higher in controls than in cases (74% versus 44%, p=0.02), while no statistical difference was found at six months (93% versus 96%). The median (IQR) time to culture conversion was shorter for controls than for cases (60 (35-89) days versus 98 (70-124) The results of this study highlight that bedaquiline-containing MDR-TB regimens appear to be associated with slower time to sputum culture conversion compared to fluoroquinolone-containing regimens. However, this difference was not significant after adjustment by other variables that may influence the outcome. The slower time to culture conversion in the bedaquiline-treated group could therefore be (at least partially) explained by the patient case mix and differences in the background regimen. Further studies, including prospective cohort studies and clinical trials, will be needed to draw higher-quality conclusions on the microbiological efficacy of bedaquiline and to relate the difference in time to culture conversion with treatment outcome.

VIII. Article 4

Rapid emergence of Mycobacterium tuberculosis bedaquiline resistance: lessons to avoid repeating past errors.

Veziris

Summary of Article 4

The development of antimicrobial resistance has invariably followed the introduction of new drugs in the history of infectious diseases, and TB represents no exception. Since the introduction of bedaquiline in clinical practice, however, only few case reports described the appearance of drug resistance to this drug. Furthermore, the resistance mutations identified in the clinical trials were surprisingly differing to those described in multiple in vitro studies. In addition, the finding of unexpected cross-resistance between clofazimine and bedaquiline, as well as the presence of mutations conferring resistance to bedaquiline among patients not exposed to this drug, and even in drug-susceptible TB strains, warranted future research.

The objective of this study was to assess the rate of resistance to bedaquiline among M. tuberculosis strains in France, and to describe the genotypic mechanisms of resistance, as well as the presence of risk factors in patients harbouring resistant strains.

A prospective surveillance study was performed at the National Reference Centre for Mycobacteria in Paris, France, including all MDR-TB strains received at the National Reference Centre between January 1 st , 2014 and December 31 st , 2015. Strains were initially tested at a concentration of 64 mg/L in Lowenstein-Jensen medium: resistant strains were further analysed by measuring bedaquiline minimum inhibitory concentration (MIC) in 7H11 medium and by sequencing of the atpE and Rv0678 genes.

Overall, 209 MDR-TB strains, including 40 XDR-TB strains, were included in the study. Among these, four (2%) had resistant strains as defined above. Two of the four patients had never received bedaquiline, nor clofazimine, before. The other two patients developed bedaquiline resistance while on MDR-TB treatment, likely because of an inadequate background regimen. In particular, one patient developed clinical failure in association to the acquisition of bedaquiline resistance and had to start an alternative salvage regimen. In all four cases, the strains showed increased MICs for bedaquiline. In three out of four cases, bedaquiline phenotypic resistance was associated with mutations in the Rv0678 gene; in one case, no resistance-conferring mutation could be identified among those described in literature to date.

The results of the study highlight a worrying rate of bedaquiline resistance among routinely-collected MDR-TB strains in France. The detection of two cases of primary drug resistance is particularly concerning. These data support routine drug resistance testing for bedaquiline on MDR-TB strains, regardless of their previous treatment exposure, to adapt treatment regimens and prevent the selection of further drug resistance. As discussed in the first part of this manuscript, the concomitant use of the two new anti-tuberculosis drugs, bedaquiline and delamanid, has been limited to little numbers of patients globally, mainly because of safety concerns. In particular, both drugs can prolong the QTcF interval, and no data from clinical trials is available on the association of the two drugs. This situation is reflected by the lack of published evidence, which is limited to only a few case series. The World Health Organization currently recommends using these drugs for a standardised duration of 24 weeks, restricting their concomitant use to patients with "no other therapeutic options". Otherwise, it is recommended to start the two drugs sequentially, after a washout period of six months when switching from bedaquiline to delamanid, due to the long half-life of bedaquiline, and of five days only when switching from delamanid to bedaquiline.

IX. Article 5

However, this recommendation is controversial as the sequential use of the drugs could increase the risk of acquisition of drug resistance and contradicts the classic principles of antituberculosis chemotherapy.

The objective of this study was to describe the safety of concomitant or sequential use of bedaquiline and delamanid as part of MDR-TB treatment regimens, with a particular focus on the length of exposure to these two drugs.

Through a multicentre case series, we included patients from three TB referral centres in the Ile-de-France region, France, and in the University Hospital of Riga, Latvia; inclusion criteria were having started treatment for culture-proven MDR-TB from January 1, 2013 to August 31, 2015, and exposure to the bedaquiline-delamanid combination for ⩾30 days.

Exposure to bedaquiline and delamanid was defined as the sum of: 1) days of concomitant treatment; and 2) days of exposure to one drug after stopping it, as defined by the World Health Organisation washout period, while taking the other one (sequential treatment).

Overall, 10 MDR-TB patients (six XDR-TB) were included. Additional resistance to rifampicin and isoniazid was found to a median of eight drugs (IQR 6-9), including resistance to bedaquiline in four cases. All patients had cavitary pulmonary TB, nine had smear-positive sputum at baseline and eight had bilateral lung involvement. Four patients were HCVinfected. Overall, six patients received concomitant bedaquiline-delamanid treatment, and four sequential treatment. Six patients received prolonged (>24 weeks) bedaquiline treatment, while nine received prolonged treatment with delamanid. Treatment duration (median, interquartile range (IQR)) was of 391 days (IQR 66-733) with bedaquiline and 532 days (IQR 286-701) with delamanid. Median exposure to the combination of bedaquiline and delamanid was 171 days (IQR 138-327); two patients receiving concomitantly the two drugs for the full treatment course. No >60 ms QTcF increase occurred, while QTcF >500 ms values occurred in two patients; however, no arrhythmia or other cardiac event was recorded. Delamanid and bedaquiline were discontinued in one patient each, due to non-cardiac adverse events. Overall, nine patients were cured, and one was lost to follow-up after eight months of treatment; at the last visit, he had a culture-negative sputum.

The results aggregated in this case series are reassuring, in particular with regards to the concerns of an additive cardiotoxicity of bedaquiline and delamanid. The prolonged blood exposure to these two drugs, even in patients treated beyond the recommended 24-week treatment courses, was associated with low rates of >500 ms QTcF prolongation and did not lead to the development of any cardiac arrhythmia. These findings, although hampered by the small sample size and by the retrospective nature of the data collection, also hint at a promising efficacy of MDR-TB regimens containing bedaquiline and delamanid.

X. Article 6

Treatment correlates of successful outcomes in pulmonary multidrug-resistant tuberculosis: an individual patient data meta-analysis.

As explained in the first part of the manuscript, the field of M/XDR-TB treatment has been rapidly evolving in the recent years due to the arrival of new anti-tuberculosis drugs on the market. However, the amount of evidence on these new drugs has been accumulating slowly due to delayed introduction in many countries, in association with regulatory hurdles, and with the overall scarce inclination to innovation in the tuberculosis field. In 2017, the WHO estimated that sufficient data would be available to evaluate the efficacy of new and repurposed drugs, at least in the few countries who pioneered their introduction, and re-assess their role in the conventional regimen. The WHO, as done previously in preparation of the 2011 recommendations, commissioned a research group of the McGill's University to perform a large meta-analysis of individual patient data of MDR-TB patients, and to present the results to the guideline writing committee. We participated to this multinational effort by sharing the data of the cohort studies presented above (Article 1, 3, and 5) and contributing to their overall interpretation.

The objective of this study was to estimate the association of favourable treatment outcomes with the use of each individual drug, independently from the others, in order to ascertain their efficacy. In addition, the impact of treatment duration and of the number of drugs included in the regimens on treatment outcomes were also assessed.

The study is a large meta-analysis of individual patient data from patients treated for MDR-TB in different countries and settings. Authors of articles published between 2009 and 2016 were contacted to retrieve outcomes, treatment regimens, and individual patient characteristics. A common database was then established and the collected variables were standardised across the different studies. Data from bedaquiline-and delamanid-treated MDR-TB patients from France were included in the analysis. Propensity score-matched generalised mixed effects logistic regression was used to assess the association of treatment with individual drugs, the number of drugs contained in the regimen, and treatment duration, with treatment success or death during treatment.

Overall, 12 030 M/XDR-TB patients from 25 countries and 50 studies were included.

The overall treatment success rate was 61%. The main results of the study were related to the assessment of treatment efficacy of individual drugs. Treatment success was associated with the use of linezolid, levofloxacin, carbapenems, moxifloxacin, bedaquiline, and clofazimine.

A reduction in mortality was associated with the use of linezolid, levofloxacin, moxifloxacin, and bedaquiline (-0•14, -0•19 to -0•10). Amikacin was associated only weakly with treatment success, while the other two second-line injectables, kanamycin and capreomycin, were associated with worse outcomes. Data on delamanid were estimated to be insufficient for a formal analysis. In a secondary analysis, the best association with favourable treatment outcomes was found with regimens including five effective drugs in the initial phase, and four effective drugs in the continuation phase. The optimal duration was unchanged with regard to previous recommendations: six to eight months of intensive phase and at a total duration of 18 to 20 months.

The results of the study are strikingly in contrast with previous knowledge on efficacy of second-line drugs for the treatment of MDR-TB, showing only modest activity of secondline injectables compared to the remarkable benefits associated with the use of last-generation fluoroquinolones, bedaquiline, and linezolid. This study represented the main source of evidence for the recent update of the WHO treatment recommendations, which revolutionised the grouping of second-line anti-TB drugs. The frame of the conventional MDR-TB treatment, including the number of drugs in the regimen and the treatment duration, were confirmed by the results of the study.

XI. Other publications

The articles described in this section, although not part of the main purpose of the thesis, share with the articles presented above the topic, MDR-TB, and the rigorous methodological approach to the research questions.

In Article A, we performed a retrospective analysis on routine data and phenotypic/genotypic DST results of MDR-TB patients notified to the CNR-MyRMA from 2010 and 2016. The aim of the study was to model the diagnostic performance of commercially-available rapid molecular tests, compared to phenotypic DST as the gold standard, to guide the choice of eligibility for the shortcourse MDR-TB treatment. This regimen is currently endorsed by the WHO for patients with resistance-conferring mutations for fluoroquinolone and second-line injectables when tested with a line-probe assay for second-line anti-tuberculosis drugs. Our study showed a suboptimal diagnostic accuracy by the line-probe assay in predicting eligibility for the shortcourse regimen, in particular due to a low specificity. As a result, almost one quarter of patients who were eligible for the shortcourse treatment using line-probe assays would receive a treatment with less than four effective drugs out of the seven composing the regimen.

In Article B, a case-case study was performed on the database of the French National Reference Center for Mycobacteria in Paris, France, to identify the characteristics associated with XDR-TB among MDR-TB cases from 1998 to 2013. Foreign birth, previous antituberculosis treatment, sputum smear positivity at baseline, and phenotypic resistance to ethambutol, were independently associated with XDR-TB. The presence of one or more of these risk factors for XDR-TB should lead to perform rapid molecular testing for resistance to second-line drugs and to adapt the empirical treatment regimen, if necessary.

In Article C, a cross-sectional online survey was designed and distributed to members of the Tuberculosis Network European Trialsgroup (TBnet), a European research network connecting clinicians and scientists interested in tuberculosis. The objective of the survey was to evaluate the clinical impact of QTc prolongation and the number of cardiac events in patients receiving bedaquiline and/or delamanid for MDR-TB treatment within the World Health Organization Europe region. Out of a total of 1044 bedaquiline-treated patients, only one cardiac event was reported (0.10%, 95% CI: 0.01%-0.63%); out of 220 delamanid-treated patients, one significant cardiac event was reported (0.45%, 95% CI: 0.02%-2.89%). Overall, the results of the study highlighted low rates of clinically relevant cardiac arrhythmias and no cardiac fatalities among patients receiving MDR-TB treatment regimens including bedaquiline and/or delamanid.

Article D is an editorial presenting a large, multicentre, retrospective cohort of MDR-TB patients treated with a bedaquiline-containing regimen in 25 centres across 15 countries.

The editorial underlined the shortcomings in the available evidence supporting the use of this drug, in particular the lack of data on the extended use of bedaquiline or on its combination with delamanid. In addition, the article highlighted the huge disproportion between the projections of MDR-TB patients in need of treatment with the new drugs and the numbers of those who actually received them, as well as the need for individualised patient management to avoid the rapid emergence of drug resistance to the new drugs.

In Article E, a mixed-methods study was performed to identify the facilitating factors and barriers to the introduction of bedaquiline for the treatment of MDR-TB across five countries (Belarus, France, Georgia, South Africa, and Swaziland) that were selected because they introduced this drug for more than 5% of total MDR-TB patients. Quantitative methods included review of public databases containing data on the number of MDR-TB cases in

Discussion

At the moment of its approval as a drug for MDR-TB treatment, bedaquiline was welcomed with both genuine hope and a considerable amount of scepticism. First of all, bedaquiline received approval by stringent regulatory authorities on the exclusive basis of Phase II clinical data. This choice, although justified by the classification of M/XDR-TB as a "neglected" disease with a poor standard-of-care treatment, has been questioned for multiple reasons. 258,295 In particular, the C208 trials were characterised by a small sample size that was powered only to show a difference in surrogate interim outcomes like time to culture conversion and culture conversion rate. Although a significant difference was eventually shown in end-of-treatment outcome in favour of the bedaquiline-containing arm with regard to the control arm, this finding was facilitated by the unexpected very low favourable outcome rate in the latter, which questioned the overall validity of the results of the study. [START_REF] Diacon | Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline[END_REF]192,195 In addition, the potential cardiotoxicity exerted by bedaquiline through the prolongation of the QT interval was arguably overemphasized and led to considerable concern in the medical community. These concerns were increased by the higher mortality which was reported in the bedaquiline arm compared to the control arm of the C208 stage 2 trial. [START_REF] Diacon | Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline[END_REF] The detailed study of the time of occurrence and of the causality of the deaths was reassuring, but could not dispel the concerns surrounding this drug.

When WHO issued the first provisional guidelines on the use of bedaquiline, these uncertainties led to restrictive recommendations, which supported the use of this drug only in cases where "no other therapeutic alternatives are available". 254 In many settings, this has led to the misconception that this drug should be reserved only for incurable cases or cases having experienced multiple treatment failures. The impossibility to provide proper monitoring, i.e. the unavailability of ECG machines, discouraged some countries from implementing bedaquiline in their national recommendations. In addition, in the aforementioned document, the WHO strongly recommended putting in place a system of pharmacovigilance for patients receiving treatment with bedaquiline. This system was supposed to support the reporting of comprehensive safety information, including all adverse events, and not only serious ones. This requirement was particularly demanding for some countries where pharmacovigilance systems where hardly in place at all and has been recurrently identified as a major hurdle in the implementation of new drugs in countries with high burden of tuberculosis. 296 Another major obstacle was the delay of national regulatory approval, which has prevented many countries, in particular among the high-burden ones, from fully integrating bedaquiline in the national tuberculosis programme. 297 Alternative mechanisms, like compassionate use and expanded access programmes, have allowed providing bedaquiline to a considerable number of patients in different countries, but often required a lengthy and cumbersome process with rigid inclusion criteria, and overall did not have the aim of replacing a formal registration of the drug. 199,208,210,[298][299][300] Moreover, the access to re-purposed drugs like clofazimine and linezolid, which represent in many cases fundamental companion drugs to bedaquiline, was also challenging in many setting. All these elements have led to relevant delays in the introduction of new drugs. In 2018, there are still many countries where bedaquiline was never used, and many others where programmatic use has not started yet (Figure 24). This is particularly striking if compared with the projected numbers of patients who would need to have access to the new drugs: according to a large multinational study, 35% of M/XDR-TB patients would benefit from receiving bedaquiline (or delamanid) as part of their treatment regimen. [START_REF] Bonnet | Identification of patients who could benefit from bedaquiline or delamanid: a multisite MDR-TB cohort study[END_REF] These estimates might be considered conservative, in particular in light of the recent change in WHO recommendations; however, up to now, less than 20% of those patients has actually received bedaquiline (or delamanid) (Figure 24). 302 This gap is a global challenge that will require both funding and political support to be bridged. 303 The limited use of bedaquiline is also reflected in the paucity of good quality real-life data on its efficacy and safety. With this regard, our studies including bedaquiline-treated patients (Publications 1, 2, 3, and 5) have contributed considerably to the overall evidence base supporting bedaquiline use. The most relevant findings were arguably those pertaining to the safety of this drug. In Publication 1, we have shown that the short-term use of bedaquiline as part of M/XDR-TB treatment was safe: although a relevant number of adverse events occurred, including hepatotoxicity in 14% and QT prolongation >500 ms in 9% of patients, no cardiac arrhythmias were recorded. Of note, only one death occurred, and it was not considered to be related to bedaquiline. The impact of these results at the moment of publication can be exemplified by the fact that this article has been cited more than 100 times so far. 298 In Publication 2, we analysed the long-term use of bedaquiline: the results were more nuanced, with severe and serious adverse events occurring in 60% and 18% of patients, respectively, and with a total of four additional deaths reported (two during treatment and two during follow-up). However, the deaths were not deemed to be related to bedaquiline, as it was the case for most of the severe/serious adverse events. Our results confirmed the potential liver toxicity of bedaquiline, in particular when given in association with other hepatotoxic drugs, and in a population with elevated incidence of Hepatitis C virus (HCV) infections. QT values above 500 ms occurred in 18% of patients, but where not associated with any cardiac event. A relevant side finding of these two publications was the safety of the association of bedaquiline with other QT-prolonging drugs, which was not tested in any of the published bedaquiline trials before. In our cohort, bedaquiline was associated with clofazimine in 44%, with moxifloxacin at 800 mg daily in 22%, with levofloxacin in 18%, and with methadone in 13% of cases. In multivariable logistic regression, however, QTcF increases of more than 60 ms from baseline and QTcF values above 500 ms were independently associated only with the co-administration of moxifloxacin at 800 mg daily. 304 Overall, our results have shown an acceptable safety profile of bedaquiline-containing MDR-TB treatment regimens. In particular, these findings, together with those provided by other cohorts, [197][198][199]202,204,305 have dissipated the initial fears related to the excess mortality reported in the bedaquiline arm of the C208 Stage 2 trial. Identifying the specific relatedness of a single drug to the adverse events experienced by patients receiving a multidrug regimen is not easy. However, the results of our studies seem to confirm that the main bedaquilinerelated toxicity is represented by liver toxicity and QT interval prolongation. The former is the most frequent adverse event during tuberculosis treatment and often requires the interruption of treatment, in particular in patients with viral hepatitis. Of note, the recent availability of highly-effective agents against HCV, with the potential of achieving sustained virologic clearance regardless of the HCV genotype, has revolutionized the treatment of this disease. While there are interactions between rifampicin and directly active HCV antivirals which contraindicate their concomitant use for drug-susceptible tuberculosis, there are no known interactions between directly active HCV antivirals and second-line anti-tuberculosis drugs, including new and re-purposed agents. Although the optimal timing of HCV treatment with regards to MDR-TB treatment is not established yet, there is growing interest in providing a concomitant treatment for both diseases, in order to prevent the development of additional liver damage and to reduce the incidence of treatment-related hepatotoxicity. 306,307 Regarding QT interval prolongation, our findings are concordant with other studies in concluding that careful ECG monitoring and aggressive management of QT prolongation and electrolyte imbalance are sufficient to avoid severe cardiac arrhythmias in most cases. 221,308 In addition, the results of Publication 2 currently represent the largest body of evidence on the safety of the use of bedaquiline beyond 24 weeks. In our cohort, no significant differences were recorded between standard and prolonged bedaquiline treatment in the incidence of total, severe and serious adverse events, including liver enzyme elevation and QT prolongation. 304 Similarly, the results presented in Publication 5 are reassuring with regards to the safety of the association of bedaquiline and delamanid, even when used together for longer than 24 weeks and up to the entire treatment duration. 309 On the other hand, our results confirmed the potential toxicity of other companion drugs, like second-line injectables and linezolid, which require intensive monitoring during treatment, and where dosing reduction strategies might be considered in order to prevent predictable adverse events. For example, some clinicians prescribe second-line injectables at thrice weekly dose once sputum culture conversion is achieved. 261 Linezolid has also been used at reduced doses in some studies, including 300 mg daily administration and 600 mg thrice weekly, with promising results. 113,[310][311][312][313] These alternative dosing strategies are currently being studied in a Phase III clinical trial (clinicaltrials.gov: NCT02754765).

The studies presented in this manuscript also contributed to shed some light on the efficacy of bedaquiline-containing regimens. In Publication 1, we reported that 97% of patients achieved culture conversion after six months of treatment. These results are remarkable and significant from a programmatic standpoint, in particular since six-month sputum culture conversion rates have been shown to be a good surrogate marker for end-oftreatment outcome. 314 In Publication 2, we have confirmed these findings by reporting favourable treatment outcome in 80% of patients at the end of MDR-TB treatment. This treatment success rate is higher than other published bedaquiline cohorts from high-burden countries, 197,198,200 but in line with reports of MDR-TB treatment in low-burden countries. 315- 317 One reason that could explain this difference, in addition to the availability of more resources, is the use of individualised, rather than standardized, treatment regimens in lowburden countries. 318 Comparable results to those of our cohort were reported in Germany in difficult-to-treat MDR-TB cases receiving bedaquiline; the final outcomes were comparable to those of drug-susceptible tuberculosis cases, confirming the hope that new and re-purposed drugs could lead to reduce the divide in treatment success between susceptible and resistant forms. 319,320 Remarkably, in our cohort two deaths occurred after the end of treatment and during the follow-up: this supports the importance to systematically implement a posttreatment follow-up period of at least 12 months and to integrate it into outcome definitions, as it has been recently proposed. 321 Publication 3 provided some insight of bedaquiline microbiological efficacy through the comparison with the most active of second-line drugs, fluoroquinolones. Our study showed no difference in culture conversion rate between bedaquiline-treated and fluoroquinolone-treated patients at six months of treatment. 322 These results hinted for the first time that bedaquiline might be promoted among the core drugs for M/XDR-TB treatment. Recently, the WHO issued a rapid communication describing the first radical change in the principles of MDR-TB treatment since its origins. According to this document, bedaquiline is now part of the group of the three drugs which should be used in the treatment of every MDR-TB case, together with last-generation fluoroquinolones and linezolid.

Clofazimine and cycloserine/terizidone are companion drugs that should be included in most treatment regimens. Delamanid is currently part of the third group of drugs, including companion drugs which should be added to reach the objective of five likely-effective drugs.

However, a re-assessment of the role of delamanid is already planned by the WHO, and it will be performed as soon as more data will become available. 284 Similarly with what has been discussed above for safety, Publications 2 and 5 also added some evidence to support the activity of prolonged bedaquiline use and of the bedaquiline-delamanid combination, respectively. 304,309 Despite these therapeutic options being reserved mostly for patients with limited treatment alternatives and advanced disease, the results were satisfying. In particular, prolonged bedaquiline treatment in our cohort allowed achieving good treatment outcomes also in patients who had delayed sputum culture conversion. The use of new drugs for extended duration and concomitantly has been advocated and is increasingly common in some settings. 293 Publication 4 represented one of the earliest reports of bedaquiline resistance and the first description of the relevant rate of acquired and primary bedaquiline resistance in France. 294 This finding was unexpected, in particular for the two cases of primary resistance, and has led the National Reference laboratory in France to adapt the routine testing on rifampicin-resistant M. tuberculosis strains. Indeed, bedaquiline phenotypic and genotypic resistance testing are now performed on all MDR-TB isolates. Recently, other studies from South Africa, Russia, and China reported the emergence of bedaquiline resistance. [276][277][278][279] In addition, an elevated number of isolates with mutations in the Rv0678 gene were found among the isolates belonging to participants of the bedaquiline C208 and C209 studies.

Interestingly, those mutations were found also in patients which were never exposed to bedaquiline nor clofazimine; in addition, the correlation between genotypic and phenotypic resistance was highly variable, and depending on the specific mutations. 275 Overall, the clinical significance of Rv0678 mutations and their impact on the efficacy of treatment with bedaquiline and clofazimine is unclear. Similarly, the clinical breakpoint for bedaquiline has not been defined yet. The results of our study advise to be cautious when treating patients harbouring isolates with increased bedaquiline MICs or with Rv0678 mutations: in those cases, bedaquiline and clofazimine might be added to the treatment regimen but should not be counted as effective.

Our studies have formed part of the evidence base supporting the revision of the WHO recommendations and have contributed in shaping them. The results of Publications 1 and 2 have been reviewed by the WHO, together with cohorts from South Africa, Armenia, and Georgia, as part of the systematic review which was performed to inform the update of the recommendations on the use of bedaquiline. Although the overall recommendations did not change, the guidelines development group downgraded the undesirable anticipated effects of bedaquiline from 'large' to 'moderate'. Furthermore, the panel reviewed the data on prolonged bedaquiline use, which was provided exclusively by the French cohort, and noted that: "Data from the French cohort allowed to assess whether the duration of bedaquiline treatment had an effect on QTc prolongation. Data seemed to indicate an absence of effect of duration of bedaquiline exposure [higher than six months] on QTc prolongation >480 ms.

However, the very limited sample size needs to be noted." 252 The findings from Publication 2 and 5 also supported the issue by the WHO of specific guidance on "off-label" use of bedaquiline and delamanid, justifying this practice when "applied by clinicians in the interest of individual patient benefit". 251 Finally, the results of the analysis of individual patient data contained in Publication 6, including the French cohorts from Publications 2 and 5, have led to the most recent change in WHO recommendations, where bedaquiline has been upgraded to Group A of the new drug classification, together with fluoroquinolones and linezolid as part of the core treatment for all M/XDR-TB cases. 284 The studies presented in this manuscript share several limitations. First, the data collection has been performed retrospectively through review of medical records. Even if performed carefully, retrospective cohort studies are prone to a series of shortcomings: some variables may not have been collected or collected partially; collected variables may be inaccurate and affected by information bias, which might be both non-differential (i.e. misclassification of events or exposure) or differential (i.e. recall bias), due to relying on individual recalling and reporting; finally, selection bias may influence the characteristics of the analysed sample. Selection bias is enhanced for Publications 1, 2, 3, and 5, where the inclusion of participants was restricted, for pragmatic reasons, to patients treated in selected referral centres in the Paris region. A comprehensive national cohort, as the one that was established in Publication 4, has the advantage of being exhaustive and therefore minimizing selection bias.

Second, the sample size of the studies is small. MDR-TB is a rare disease in France and the number of cases is limited. In addition, MDR-TB patients are spread to a big number of centres throughout the country, some of them treating only one or two patients per year. In the context of retrospective, ad hoc data collection and analysis, it is therefore difficult to include all patients at the national level. In addition, as underlined above, the distribution of patients among centres with very different level of specialisation and expertise increases the heterogeneity of the patient management and monitoring.

Third, the cohort studies (Publications 1, 2, and 5) did not have a control group. The absence of a control group complicates the interpretation of the contribution of a single drug to the efficacy and safety of treatment regimens, which is already confounded by the use of non-standardized drug combinations which change multiple times during treatment. In Publication 3, for example, the availability of a control group has allowed to limit the confounders when comparing the activity of two drugs (bedaquiline and fluoroquinolones).

Fourth, all the studies were observational. Observational studies are easier and quicker to conduct, allow the use of routinely-collected data, require less funding, and are more adapted to settings with multiple centres with a small number of patients. However, the quality of evidence provided by observational studies is inferior to that of interventional studies; although statistical methods can adjust for measured confounders, they cannot compensate for other unmeasured or unknown confounders.

Perspectives

The studies presented in this manuscript have been performed pragmatically to exploit at the best the available routinely-collected data in a small number of centres which are specialised in the management of a rare diseases, M/XDR-TB. A few conditions have contributed to increase the interest of the results of such studies: the fact that France had early access first to bedaquiline, also linked to the contribution of the National Reference Center for Mycobacteria in the last stages of development of this drug, and subsequently to delamanid; the presence of a national Consilium supervising the management of complicated drugresistant tuberculosis cases, which allowed for standardisation of patient management and quick increase of the expertise in the use of new anti-tuberculosis drugs; 323 the flexibility of the French national drug agency, which allowed for the "off-label" use of bedaquiline and delamanid whenever the clinicians deemed it necessary; the availability of all resources needed to provide an optimal management of M/XDR-TB cases, including companion drugs, implantable catheters, drugs for the treatment of adverse events, advanced diagnostics, and therapeutic monitoring of the blood levels of the administered drugs. However, these studies present multiple methodological flaws, which were described in the last part of the Discussion.

Since a few years, the National Reference Centre for Mycobacteria is developing the project of a national registry of MDR-TB cases. This tool, funded by the French Direction Générale de la Santé (Ministry of Health) and supported by Santé Publique France (the national Public Health agency), will be structured as a web-based, online only, shared patient file, with multiple objectives: to improve and standardise the management and follow-up of M/XDR-TB cases across all centres seeing these patients at a national level; to facilitate the exchange of information and continuity of care across different health centres, a particularly challenging task considering the high mobility of these patients and the length of treatment; to assure the availability of high-quality surveillance data, in particular with regard to treatment outcomes, to be shared with national and international organisms (i.e. Santé Publique France, WHO, European Centre for Disease Prevention and Control); improve the coverage and efficacy of the detection, diagnosis, and management of contacts of M/XDR-TB cases, as well as providing data on their follow-up. In addition, the national M/XDR-TB registry will provide an excellent platform to establish a comprehensive, prospective national cohort that could attain multiple research goals. I have been involved in the conceptualization and creation of the national M/XDR-TB registry since the beginning, as part of my research project for a Master's degree in Epidemiology and Public Health. In the next months, I will support the gradual implementation of the registry, through a first pilot period in the same reference centres of the Paris region which were involved in the studies presented in this manuscript, followed by a second period of national roll-out. This project has a high level of priority for the National Reference Centre for Mycobacteria, as it has the potential of improving the quality of patient management, perfectioning the surveillance of drugresistance in France, and becoming a platform for collaborative research.

Indeed, once the prospective M/XDR-TB cohort will be established, several substudies could be easily nested, exploiting the availability of a detailed database with standardized variables. First, a comparison could be performed between patients receiving new drugs and those who did not, which would constitute the control group. Statistical methods, from logistic regression to G-formula based techniques, 324,325 allow to adjust for the different characteristics of such two non-randomized groups and to minimize the strong selection bias (i.e. the fact that new drugs are usually given to patients with more advanced disease and harbouring M. tuberculosis isolates with additional resistance to more drugs), provided that the sample size is big enough and that most of the variables which could potentially impact on the outcome are measured. 326,327 The main objectives of this study would be assessing interim (i.e. time to sputum culture conversion) and final treatment outcomes, including a post-treatment follow-up period, as well as safety and the costs for the healthcare system. In addition, ad hoc cohorts of drug-susceptible and isoniazid-resistant tuberculosis cases could be constituted to perform case-control studies across subgroups with different drug resistance profiles, as partially performed in previous studies. 320,328 Second, the cohort would allow to analyse the impact of performing routine measurement of blood drug levels in M/XDR-TB patients. Blood drug concentrations have been shown to be correlated to toxicity and to treatment outcome. [329][330][331] Furthermore, due to individual metabolism characteristics and drug-drug interactions, blood drug levels of firstand second-line anti-tuberculosis drugs are often excessively low. 332,333 In addition, therapeutic drug monitoring may have a role, at least with regard to M/XDR-TB treatment, also in low-resource setting, following technical improvements that facilitate the collection and storage of samples like the use of dried blood spots. [334][335][336] This study would analyse the relationship between drug concentrations and safety/efficacy outcomes, allowing to establish a) the drugs for which therapeutic drug monitoring should be routinely performed, b) the best monitoring frequency, and c) the concentration range which is associated with the best efficacy and safety.

Third, another objective would be to assess the impact of the implementation of new diagnostic tests on the management of M/XDR-TB. In the last years, the development of rapid molecular-based tests like Xpert ® MTB/RIF and line-probe assays like Hain ® first-and second-line tests has revolutionized the diagnostic algorithm of drug-resistant tuberculosis in both high-and low-burden settings and provided a faster alternative to conventional culturebased testing. [337][338][339][340][341] The Hain ® second-line test, which allows for rapid detection of resistance to fluoroquinolone and second-line injectables, is of paramount importance to define the first empiric treatment approach of rifampicin-resistant tuberculosis cases. 345 This test is implemented since a few years at the National Reference laboratory in Paris. A study could be performed to assess the impact of Hain ® second-line, including both the first and second version of the test, 346 on the time to start an adequate treatment regimen and on interim and final treatment outcomes of M/XDR-TB patients, also taking into account the use of conventional sequencing. In the future, the most exciting developments in tuberculosis diagnostics are most likely going to be based on whole genome sequencing: the prospective M/XDR-TB cohort will represent an ideal platform to analyse the added value of this technique and to compare it to rapid molecular testing and conventional targeted sequencing. 347,348 Fourth, a sub-study testing biomarkers could be easily included in a subgroup or in the whole cohort of M/XDR-TB patients. With the availability of new and re-purposed drugs, the treatment of M/XDR-TB is rapidly improving, as shown by the last recommendations by the WHO. The biggest challenge currently appears to be the individualization and overall shortening of treatment. A series of biomarkers are being studied in different aspects of tuberculosis management, 349 including for example the prediction of risk of developing active tuberculosis among persons with latent tuberculosis infection. 350,351 However, the biomarkers which could arguably have the biggest clinical impact are those capable of predicting the response to anti-tuberculosis treatment and guide the treatment interruption. So far, only sputum culture status has shown some value as a predictor of treatment response. [352][353][354] Recently, other biomarkers have emerged as very promising candidates, although they were only tested in drug-susceptible patients. 355,356 A study could be performed to analyse their performance in patients receiving M/XDR-TB treatment, first as a pilot study and subsequently in a cohort of patients where the treatment duration would be guided by the results of the biomarker.

Well-planned and performed observational studies can provide high-quality evidence and inform international policies. This principle has been recently accepted also in the field of drug-resistant tuberculosis treatment, when the WHO has recommended in 2016 a shorter MDR-TB regimen on the basis of multiple observational cohort studies. [START_REF] Aung | Successful '9-month Bangladesh regimen' for multidrug-resistant tuberculosis among over 500 consecutive patients[END_REF][START_REF] Piubello | High cure rate with standardised short-course multidrug-resistant tuberculosis treatment in Niger: no relapses[END_REF][START_REF] Trébucq | Treatment outcome with a short multidrug-resistant tuberculosis regimen in nine African countries[END_REF]285 However, the risks of such an approach have been demonstrated in the following year, when a Phase III trial has failed to show non-inferiority of the shorter MDR-TB regimen with regard to the conventional MDR-TB regimen. 357 Randomized, controlled clinical trials still represent the most reliable source of evidence, and particularly so in the field of chemotherapy. ........................................................................................................................... Figure 14. Time to sputum culture conversion, trial C208 Stage 2. ........................................ pretomanid. .............................................................................................................................. Figure 20. Sputum culture conversion rates of participants of Trial 204. ............................... Figure 21. Schematic of conventional MDR-TB regimen and delamanid trials. ..................... Figure 22. Structure and patient disposition of delamanid trials. ........................................... Figure 23. Pipeline of clinical and preclinical development of new anti-tuberculosis drugs. ........................................................................................................................................ Figure 24. Global overview of the use of bedaquiline and delamanid. ................................. Table 1. Drug-drug interactions of bedaquiline and cytochrome P450 inducers/inhibitors. .................................................................................................................. Table 2. Drug-drug interactions of bedaquiline and antiretrovirals. ...................................... Table 3. Characteristics of the study participants of the three bedaquiline Phase IIb trials. ........................................................................................................................................ Table 4. Efficacy outcomes of the three bedaquiline Phase IIb trials...................................... Table 5. Summary of observational studies describing MDR-TB patients treated with bedaquiline. .............................................................................................................................. Table 6. Summary of main safety findings in the three bedaquiline Phase IIb trials. ............. Table 7. Drug-drug interactions of delamanid and cytochrome P450 inducers/inhibitors. .... Table 8. Drug-drug interactions of delamanid and antiretrovirals. ........................................ Table 9. Efficacy outcomes of delamanid trials. ...................................................................... Table 10. Summary of the characteristics of the observational studies describing MDR-TB treatment with delamanid. .................................................................................................. Table 11. Main safety findings in the delamanid Phase IIb and Phase III trials..................... Table 12. Grouping of anti-tuberculosis drugs according to 2018 World Health Organization recommendations. .............................................................................................. Table 13. Characteristics of the main clinical trials testing new and shorter combinations for fluoroquinolone-susceptible MDR-TB. ............................................................................... Table 14. Characteristics of the main clinical trials testing new and shorter combinations for fluoroquinolone-resistant MDR-TB. ...................................................................................

TABLE OF FIGURES

TABLE OF TABLES

RESUME EN FRANÇAIS Introduction

Revue systématique de la littérature

Une revue systématique de la littérature a été effectuée sur MEDLINE pour les articles publiés jusqu'au 30 Juin 2018 en incluant les mots-clés suivants : "bedaquiline" AND "tuberculosis", "TMC207" AND "tuberculosis", "delamanid" AND "tuberculosis", "new drugs" AND "tuberculosis". Les titres et les résumés des articles ont été ensuite revus systématiquement et pour les études qui rapportaient des résultats originaux, les manuscrits entiers ont été analysés. De plus, 11 articles de revue et quatre revues systématiques ont été identifiés, et analysés pour consolider l'exhaustivité de la stratégie de recherche. Le suivi de seulement six mois à partir du début du traitement n'était néanmoins pas suffisant pour une évaluation complète de l'intérêt de ce nouvel antibiotique.

Bédaquiline

Le second travail sur la cohorte est la suite logique du premier, avec l'évaluation du devenir des patients (succès/échec) et de la tolérance du traitement avec un suivi prolongé jusqu'à 24 mois après la fin du traitement, soit 42 mois au total après le début du traitement.

Ce travail a aussi permis de comparer les malades traités par bédaquiline pour une durée standard (<190 jours) ou une durée prolongée (≥190 jours). Le taux d'évènements indésirables était élevé, mais en grande majorité en lien avec les autres antituberculeux comme le linézolide. L'administration prolongée de la bédaquiline n'était pas associée à un surcroit de toxicité, ni à des taux plus élevés de prolongation de l'intervalle QT. Ces résultats supportent l'utilisation prolongée de la bédaquiline, en prenant en compte le fait que les patients ayant reçu un traitement prolongé ont eu le même taux de succès thérapeutique que les patients ayant reçu le traitement par bédaquiline pour une durée « standard », alors que dans les patients du groupe bédaquiline prolongée avaient des caractéristiques suggérant une complexité plus importante du traitement et que le temps de conversion des cultures était plus long. Ce travail était, au moment de la revue de la littérature, la cohorte la plus importante de malades ayant reçu un traitement prolongé par bédaquiline. Les études présentés dans ce travail pourraient déboucher vers plusieurs filières de recherche. Il est d'abord prévu de mettre en place une cohorte observationnelle prospective, par le biais d'un registre informatisée, de tous les cas de tuberculose à bacilles M/XDR au niveau national. Cette cohorte, qui combinera des objectifs de surveillance, amélioration de la prise en charge et de la communication entre médecins, et de recherche, pourra être exploité pour plusieurs études, comme par exemple : 1) une comparaison entre patients traités et pas traités par nouvelles molécules, 2) une analyse de l'impact des concentrations plasmatiques sur le devenir des malades, 3) une évaluation de l'apport des nouveaux tests diagnostiques rapides, et 4) une analyse des performance de nouveaux marqueurs pour la prédiction de l'issue du traitement. Mots clés : Tuberculose ; multirésistance ; ultrarésistance ; bédaquiline ; delamanide ; traitement.

Figure 2 .

 2 Figure 2. Incidence, notification rates, and mortality rates in France (1972-2016).

  Number of notified cases (light blue columns), rate of new tuberculosis cases per 100 000 persons (dark blue line), and mortality rate per 100 000 persons (red line) in France from 1972 to 2016. Source: Santé Publique France, 2017.

Figure 3 .

 3 Figure 3. Different mycobacterial populations and response to first-line anti-tuberculosis drugs.

Figure 4 .

 4 Figure 4. Global rates of rifampicin-resistant/multidrug-resistant tuberculosis.

Figure 5 .

 5 Figure 5. Development of drug resistance in Mycobacterium tuberculosis.

Figure 6 .

 6 Figure 6. Selection of drug resistance in Mycobacterium tuberculosis.

Figure 7 .

 7 Figure 7. Mechanism of action of anti-mycobacterial drugs on Mycobacterium tuberculosis.

Figure 8 .

 8 Figure 8. Number of published articles on new anti-tuberculosis drugs, per year.

Figure 9 .

 9 Figure 9. Chemical structure of bedaquiline.Source:Andries et al. 131

Figure 10 .

 10 Figure 10. Morphologic structure of ATP synthase. The structure of ATP synthase includes F0 and F1 components and their subunits (image on the left), and partial surface representation of F1 subunit (light grey: subunit alpha; dark grey: subunits beta; magenta: subunit gamma; pink: subunit delta; violet: subunit epsilon) (image on the right). Source: www.atpsynthase.info.

Figure 11 .

 11 Figure 11. Seven-days early bactericidal activity of bedaquiline.Seven-day early bactericidal activity of bedaquiline (TMC207 in the figure) at different doses between 25 and 400 mg daily, compared to rifampin and isoniazid. Source:Rustomjee et al. 189

Figure 12 .

 12 Figure 12. 14-days early bactericidal activity of bedaquiline.14-days early bactericidal activity of bedaquiline (BDQ in the figure) at different doses between 100and 400 mg daily, preceded by a loading dose, and compared to the standard anti-tuberculosis treatment (Rifafour in the figure). Source:Diacon et al. 190

Figure 13 .

 13 Figure 13. Time to sputum culture conversion of bedaquiline and placebo arm, trial C208 Stage 1.

Figure 14 . 66 Figure 15 .

 146615 Figure 14. Time to sputum culture conversion, trial C208 Stage 2. Kaplan-Mayer curve showing the time to culture conversion of sputum samples of MDR-TB patients receiving either bedaquiline (blue line) or placebo (yellow line) plus optimised background regimen in study C208 Stage 2. Source: Diacon et al.[START_REF] Diacon | Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline[END_REF]

  mITT = modified intention-to-treat; TCC = time to sputum culture conversion; Cult Conv = rate of culture conversion at the endpoint; Bdq = bedaquiline.Study C209 is an open-label, single-arm, Phase IIb clinical trial that aimed to confirm the safety of bedaquiline. Overall, 233 patients with pulmonary MDR-TB were included and treated with bedaquiline (400 mg daily for 2 weeks, followed by 200 mg three times a week for 22 weeks) plus an optimised background regimen. The total follow-up was 120 weeks. In the modified intention-to-treat population (n=205), culture conversion was 72.2% at 120 weeks. The median time to culture conversion was 57 days. At study completion, 125 patients (61.0%) were cured, 3 (1.5%) completed treatment, 32 (15.6%) failed treatment, 31 (15.1%) defaulted, and 14 (6.8%) died according to WHO outcome definitions for

  Pts = patients; AE = adverse event; SAE = serious adverse event; Bdq = bedaquiline; Plac = placebo; TB = tuberculosis.

Figure 16 .

 16 Figure 16. Chemical structure of nitroimidazoles. Chemical structure of delamanid (OPC-67683 in the figure), pretomanid (PA-824 in the figure) and another 4-nitroimidazole compound that was not further developed (CGI-17341). Source: Matsumoto et al. 222

  Nitro-heterocyclic compounds have shown pharmacological properties in different fields of chemotherapy and have been extensively studied in drug development.224 A wellknown antibacterial belonging to this class is metronidazole, which is one of the most commonly used drugs to treat infections by anaerobic bacteria. The 4-nitrodihydroimidazooxazoles (4-nitroimidazoles) like pretomanid and delamanid, in particular, exhibit interesting anti-tuberculosis properties. The mechanism of action of 4-nitroimidazoles involves the inhibition of the synthesis of the mycobacterial cell wall components. In particular, delamanid inhibits the synthesis of methoxy-and keto-mycolic acids, but not that of α-mycolic acids. This leads to both preventing mycobacterial replication and enhancing drug penetration. The 4-nitroimidazole compounds have little or no activity on other Grampositive or Gram-negative bacteria.The growing interest in the 4-nitroimidazoles is testified by the increasing number of studies assessing other compounds belonging to this class, including TBA-354.223,[225][226][227] So far, however, none of these other molecules (except for pretomanid) has advanced to the stage of clinical testing.

Figure 17 .

 17 Figure 17. 14-days early bactericidal activity of delamanid.14-days early bactericidal activity of delamanid at different doses between 100 and 400 mg daily, compared to the standard first-line anti-tuberculosis regimen (HRZE in the figure). Source: Diacon et al. 238

Figure 18 .

 18 Figure 18. 14-days early bactericidal activity of drug combinations including bedaquiline and/or delamanid. 14-days early bactericidal activity of different combinations of bedaquiline (B in the figure), pretomanid (Pa in the figure), pyrazinamide (Z in the figure), clofazimine (C in the figure), and of the standard firstline anti-tuberculosis regimen (Rifafour e-275 in the figure). Source:Diacon et al. 194

4 )

 4 bedaquiline-pretomanid-clofazimine; 5) clofazimine alone; 6) pyrazinamide alone; 7) standard first-line anti-tuberculosis treatment. The highest EBA was recorded in the bedaquiline-pretomanid-pyrazinamide arm, which was superior to the standard first-line antituberculosis treatment arm (Figure18).194

Figure 19 .

 19 Figure 19. 56-days early bactericidal activity of drug combinations including pretomanid. 56-days early bactericidal activity of different combinations of moxifloxacin (M in the figure), pretomanid at 100 mg or 200 mg daily (Pa100 and Pa200 in the figure, respectively), pyrazinamide (Z in the figure), and of the standard first-line anti-tuberculosis regimen (HRZE in the figure) in drugsusceptible tuberculosis patients, and of the association of moxifloxacin, pretomanid at 200 mg daily, and pyrazinamide in drug-resistant tuberculosis patients. Source:Dawson et al. 239

Figure 20 .

 20 Figure 20. Sputum culture conversion rates of participants of Trial 204.Sputum culture conversion rates on liquid (above) and solid culture media (below) for MDR-TB patients treated in Trial 204 with optimized background regimen plus either placebo (yellow), delamanid at 100 mg twice daily, or delamanid 200 mg twice daily (both green). Source: Gler et al.[START_REF] Gler | Delamanid for multidrug-resistant pulmonary tuberculosis[END_REF]

Figure 21 .

 21 Figure 21. Schematic of conventional MDR-TB regimen and delamanid trials.MDR-TB treatment regimen according to the WHO recommendations (=WHO OBR) (a) and presentation of the delamanid development program including Trial 204, Trial 208, and Observational Study 116 (b). Source: Skripconoka et al. 67 SCC = sputum culture conversion.

Figure 22 .

 22 Figure 22. Structure and patient disposition of delamanid trials.Overall structure and patient recruitment disposition of the delamanid development program includingTrial204, Trial 208, and Observational Study 116 (b). Source: Skripconoka et al.[START_REF] Gler | Delamanid for multidrug-resistant pulmonary tuberculosis[END_REF] DLM = delamanid.

  partially expanded over time (i.e. approval by the WHO of the pediatric use of delamanid). This situation, associated with the lack of effective drugs for M/XDR-TB, has pushed clinicians to use bedaquiline and delamanid in situations that partially contrast the authorised indications (i.e. pediatric populations, extrapulmonary tuberculosis, extended treatment duration) or which expand them (combination of bedaquiline and delamanid, use in pregnant women). The first category falls under the term of "off-label use", defined as "the use of a pharmaceutical agent for an unapproved indication or in an unapproved age group, different dosage, duration or route of administration".251 Off-label use is quite common in the medical practice, and in particular in the field of M/XDR-TB treatment, where most of the drugs, including fluoroquinolones, second-line injectables, linezolid, clofazimine, and others, are actually not approved for the use in tuberculosis.

  represent a valid option to treat a deadly disease like MDR-TB. A cohort study describing the off-label use of bedaquiline in 27 children (mostly adolescents) from different countries showed an overall good safety, apart from a few cases of QT prolongation. Information from this study and from unpublished data of adolescents treated with bedaquiline in South Africa led the WHO to acknowledge the presence of evidence, although of low quality, supporting the use of bedaquiline in adolescents. 252 Since its approval, delamanid has been the drug of choice in paediatric MDR-TB patients that needed a new anti-tuberculosis drug. This is mainly the consequence of the engagement of the manufacturer to perform studies on the pharmacokinetics and tolerability of delamanid in children. In 2016, the WHO issued specific recommendations expanding the indications of delamanid to children from 6 years of age. The recommended dose is 50 mg twice daily for 6 months in children from 6 to 11 years of age, and 100 mg twice daily for 6 months in adolescents from 12 years of age. 253 This change in the guidelines was supported by a cohort of children treated with delamanid for M/XDR-TB in different countries, showing

  Bedaquiline resistance emerges through the selection of spontaneous chromosomal mutants. Initial studies of in vitro selection of M. tuberculosis mutants resistant to bedaquiline showed that the mutation rate for bedaquiline was comparable to the one for rifampicin (approximately one mutant every 10 7 bacteria) and was independent from the drug susceptibility to other anti-tuberculosis drugs. Bedaquiline-resistant isolates harboured mutations in the atpE gene, 131 a highly-conserved gene encoding for the subunit c of the mycobacterial ATP synthase.171 Mutations in atpE are associated with 8-to 133-fold increase in bedaquiline MICs, defined as high-level resistance. Conversely, in a subsequent study, 38 out of 53 bedaquiline-resistant tuberculosis isolates did not show any mutation in the atpE gene, nor in other known genes coding for components of the mycobacterial ATP synthase, suggesting the presence of alternative resistance mechanisms.269 In 2014, a clinical case was published describing for the first time the acquisition of bedaquiline drug resistance during treatment, with the simultaneous appearance of resistance to clofazimine in absence of any exposure to this drug. Drug resistance to bedaquiline and clofazimine appeared to be linked to a mutation in the rv0678 gene. 217 Rv0678 codes for a transcriptional repressor that binds a sequence located in the intergenic region between rv0678 and the neighbouring genes, mmpS5 and mmpL5. Mutations in rv0678 were found to lead to depression of this locus with subsequent transcriptional up-regulation of all three genes (rv0678, mmpS5, and mmpL5) and increased production of proteins belonging to the MmpS5-MmpL5 family, 270 which are multisubstrate efflux pumps that are also mediate resistance to azoles in M. tuberculosis.271 Overall, mutations in rv0678 lead to a 2-to 8-fold increase in bedaquiline MICs and to a 2-to 4-fold increase in clofazimine MICs; the clinical impact of this intermediate-level resistance remains to be established.196 The efflux pump-mediated mechanism of cross resistance between bedaquiline and clofazimine has been confirmed by the finding that the administration of an efflux pump inhibitor like verapamil reduced in vitro the MICs to both drugs. However, this reduction was observed also in susceptible strains, suggesting that efflux-based mechanisms are implied in intrinsic resistance to bedaquiline, in addition to a possible role in acquired resistance.272 Moreover, verapamil failed to increase the bactericidal effect of bedaquiline in mice and was unable to reverse efflux-based resistance in vivo.273 Recently, mutations in pepQ gene have been identified as sources of resistance mediated by efflux pump systems to both clofazimine and bedaquiline. Mutations in pepQ lead to a 4-fold increase in bedaquiline and clofazimine MICs.274

  manufacturer engaged with the FDA to perform a Phase III trial to increase the amount of evidence on bedaquiline.220 Bedaquiline has received marketing approval by the EMA in 2014. Delamanid has been approved by the EMA in 2014, based on the review of results from Trials 204, 208, and 116. FDA approval is still pending and a request has been filed with the results of Trial 213.282

Figure 23 .

 23 Figure 23. Pipeline of clinical and preclinical development of new anti-tuberculosis drugs.

Table 13 .Table 14 .

 1314 biosynthesis of the cell wall. Although officially at Phase II of clinical development due to a small study that has been performed in the Russian Federation and whose results are still unpublished, the drug is currently being tested in a Phase I study in Switzerland. In conclusion, there are currently no innovant anti-tuberculosis drugs in Phase II or III of clinical development. Clinicians treating drug-resistant tuberculosis cannot therefore expect any new breakthrough in terms of new chemotherapy agents, at least for the next few years. Characteristics of the main clinical trials testing new and shorter combinations for fluoroquinolone-susceptible MDR-TB. = fluoroquinolone-susceptible; SLI-S = susceptible to second-line injectables; MDR-TB = multidrugresistant tuberculosis; DS-TB = drug-susceptible tuberculosis; DR-TB = drug-resistant tuberculosis; Bdq = bedaquiline; Dlm = delamanid; Pa = pretomanid; HRZE = isoniazid, rifampicin, pyrazinamide, and ethambutol; SoC = standard-of-care; mo = months; TTP = time to culture positivity; TCC = time to culture conversion. Characteristics of the main clinical trials testing new and shorter combinations for fluoroquinolone-resistant MDR-TB. FQ-R = fluoroquinolone-resistant; MDR-TB = multidrug-resistant tuberculosis; Bdq = bedaquiline; Dlm = delamanid; Pa = pretomanid; SoC = standard-of-care; mo = months. Conversely, innovation is expected to come in the next years in the form of the identification of new, shorter MDR-TB treatment regimens including new and re-purposed drugs. A relevant number of Phase II and Phase III clinical trial are ongoing or starting soon and are summarised in Table

  Guglielmetti L, Le Dû D, Veziris N, Caumes E, Marigot-Outtandy D, Yazdanpanah Y, Robert J, Fréchet-Jachym M, for the MDR-TB management group of the French National Reference Center for Mycobacteria and the physicians of the French MDR-TB cohort. European Respiratory Journal 2016; 48:582-5.

  N, Bernard C, Guglielmetti L, Le Du D, Marigot-Outtandy D, Jaspard M, Caumes E, Lerat I, Rioux C, Yazdanpanah Y, Tiotiu A, Lemaitre N, Brossier F, Jarlier V, Robert J, Sougakoff W, Aubry A, on behalf of the CNR MyRMA and the tuberculosis Consilium of the CNR MyRMA. European Respiratory Journal 2017; 49:1601719.

  Barkane L, Le Dû D, Marigot-Outtandy D, Robert J, Veziris N, Yazdanpanah Y, Kuksa L, Caumes E, Fréchet-Jachym M. European Respiratory Journal 2018; 51:1702550.

  2014/2015 in each country, as well as the number of patients started on bedaquilinecontaining regimens in the same years. Qualitative methods included review of program documents, interviews with directors of the national TB programs, interviews with other key stakeholders working on bedaquiline introduction, and direct observation of meetings on and activities involving bedaquiline introduction. Common facilitating factors observed by the national TB programs of these countries included support from external implementing partners, experience with compassionate use and/or expanded access programs, and adequate national or donor-supported budgets. Common barriers included having no access to companion drugs, the restriction of bedaquiline to hospitalised patients, and the development of pharmacovigilance systems.The objective of Article F was to quantify the number of eligible patients for the shorter MDR-TB regimen across Europe. The standardized shorter MDR-TB treatment has been approved by the World Health Organisation in 2016 for patients without documented or likely resistance to any of the drugs included medicines in the regimen, with the exception of isoniazid. We performed a retrospective evaluation of consecutive MDR-TB strains in three referral centres in Austria, France, and Germany, from the national Portuguese MDR-TB database, and from the TBnet MDR-TB cohort database, the latter including data from individual patients from 16 countries. Overall, out of 612 assessable strains, only 48(7.8%) had full susceptibility to the drugs proposed in the shorter MDR-TB regimen. These results suggest that the impact of the shorter MDR-TB regimen in Europe might be limited and underline the importance of performing full genotypic and phenotypic drug susceptibility testing when screening MDR-TB patients for the shorter regimen in this region.

Figure 24 .

 24 Figure 24. Global overview of the use of bedaquiline and delamanid. Above: countries which have started implementing bedaquiline in programmatic use (in blue) and outside programmatic use only (in green); below: estimated global number of patients having received bedaquiline (blue), delamanid (red), compared to the number of patients who would need one of those new drugs according to conservative estimations. 301 Source: DR-TB STAT, update June 2018 (http://drtb-stat.org/global-snapshot/).

  Affiliated with Médecins Sans Frontières (MSF), I have participated since 2015 and in different positions to the endTB (expand new drug markets for TB) project (www.endtb.org), an ambitious partnership between three non-governmental organizations: MSF, Partners In Health, and Interactive Research and Development. This project, funded by UNITAID, has multiple objectives: a) expand the use of new anti-tuberculosis drugs involving 17 countries and at least 2600 patients, and study the efficacy and safety results through a prospective cohort study; b) perform two clinical trials to identify simpler, less toxic, more effective treatment regimens to treat fluoroquinolone-susceptible (endTB trial) and fluoroquinoloneresistant MDR-TB (endTB-Q trial); c) use this evidence to advocate for registration and implementation of new drugs globally; and d) share the findings with international stakeholders and policy makers to ultimately influence treatment recommendations. I am currently co-Principal Investigator of the endTB (clinicaltrials.gov: NCT02754765) and endTB-Q (recently approved, not registered yet) clinical trials, both Phase III randomized, controlled clinical trials comparing multiple experimental arms to the standard-of-care MDR-TB regimen. The endTB trial has started enrolling in early 2017, while enrolment for endTB-Q is planned to start in the first quarter of 2019; the final results for the two trials are expected between 2021 and 2022 and will hopefully shape the state of the art of treatment of M/XDR-TB in the future.

Figure 1 .

 1 Figure 1. Evolutionary diagram of Mycobacterium tuberculosis. .............................................

Figure 2 .

 2 Figure 2. Incidence, notification rates, and mortality rates in France (1972-2016). ................

Figure 3 .

 3 Figure 3. Different mycobacterial populations and response to first-line anti-tuberculosis drugs. ..........................................................................................................................................

Figure 4 .

 4 Figure 4. Global rates of rifampicin-resistant/multidrug-resistant tuberculosis. .....................

Figure 5 .

 5 Figure 5. Development of drug resistance in Mycobacterium tuberculosis. ...........................

Figure 6 .

 6 Figure 6. Selection of drug resistance in Mycobacterium tuberculosis. ..................................

Figure 7 .

 7 Figure 7. Mechanism of action of anti-mycobacterial drugs on Mycobacterium tuberculosis. .............................................................................................................................

Figure 8 .

 8 Figure 8. Number of published articles on new anti-tuberculosis drugs, per year. ................

Figure 9 .

 9 Figure 9. Chemical structure of bedaquiline. ..........................................................................

Figure 10 .

 10 Figure 10. Morphologic structure of ATP synthase. ................................................................

Figure 11 .

 11 Figure 11. Seven-days early bactericidal activity of bedaquiline. ...........................................

Figure 12 .

 12 Figure 12. 14-days early bactericidal activity of bedaquiline. ................................................

Figure 13 .

 13 Figure 13. Time to sputum culture conversion of bedaquiline and placebo arm, trial C208Stage 1. ...........................................................................................................................

Figure 15 .

 15 Figure 15. Treatment outcomes of placebo and bedaquiline arm, study C208 Stage 2. .........

Figure 16 .

 16 Figure 16. Chemical structure of nitroimidazoles. ..................................................................

Figure 17 .

 17 Figure 17. 14-days early bactericidal activity of delamanid. ..................................................

Figure 18 .

 18 Figure 18. 14-days early bactericidal activity of drug combinations including bedaquiline and/or delamanid. ................................................................................................

Figure 19 .

 19 Figure 19. 56-days early bactericidal activity of drug combinations including

  La tuberculose, qui est une infection à mycobactéries du complexe Mycobacterium tuberculosis, est la 9 ème cause de mortalité dans le monde. Selon l'Organisation Mondiale de la Santé (OMS), en 2016 il y a eu environ 10.4 million des nouveaux cas dans le monde, soit un taux d'incidence moyen de 140 malades par 100 000 habitants. Toutefois, cette incidence est en diminution, et cela plus particulièrement dans la région européenne de l'OMS.Néanmoins, les progrès ont été ralentis par plusieurs facteurs, notamment l'épidémie de VIH et la diffusion de souches de tuberculose à bacilles résistants aux antibiotiques. Le nombre réel de cas à bacilles multirésistants aux antituberculeux (multidrug-resistant ou MDR), définies par une résistance à la rifampicine et l'isoniazide, et des cas à bacilles ultrarésistants (extensively drug-resistant ou XDR), définis comme des cas MDR avec une résistance additionnelle à une fluoroquinolone et à un médicament injectable de deuxième ligne, est difficile à évaluer au niveau mondial, en raison de l'absence de laboratoires capables d'effectuer des tests de sensibilité aux antituberculeux dans de nombreux pays. L'OMS estime qu'en 2016 il y a eu environ 490 000 nouveaux cas de tuberculose MDR, avec environ 240 000 décès associés aux cas MDR ou monorésistants à la rifampicine. En France, l'incidence annuelle de la tuberculose à bacilles M/XDR a été historiquement assez stable autour de 50 cas par an ; ces dernières années, la France a connu une augmentation du nombre des cas MDR, en particulier liée aux flux migratoires de l'Europe de l'est, du Caucase, et de l'Asie Centrale. Le traitement recommandé par l'OMS jusqu'à 2018 pour les cas de tuberculose MDR est au moins trois fois plus long que pour une tuberculose à bacilles sensibles et est souvent associé à de nombreux effets secondaires. La durée totale du traitement est de 18 à 24 mois, avec une phase intensive de 6-9 mois comprenant un aminoside, suivie par une phase d'entretien totalement orale. Le traitement est doit contenir quatre antituberculeux actifs en dehors du pyrazinamide. Le taux mondial de succès thérapeutique n'atteint pas 60 % dans plusieurs méta-analyses et dans le rapport global de l'OMS en 2017. Ce taux est encore moindre pour les cas de tuberculose XDR. En France, il n'y a pas de données nationales systématiquement collectées sur le devenir des cas MDR et XDR. La découverte du premier médicament efficace contre les bacilles tuberculeux, la streptomycine, date de 1943. Dès les premiers patients traités, on a constaté l'émergence d'une résistance à cette molécule. Ceci a mené au concept de polychimiothérapie pour prévenir la sélection des mutants résistants. Le traitement devait être très long pour éviter les rechutes et était initialement administré pendant 18 mois. La chirurgie, initialement le seul traitement avant les antibiotiques, jouait encore un rôle important dans la majorité des cas, pour être dans un second temps limitée aux cas avec des bacilles résistants aux antituberculeux. Le traitement antituberculeux standard (ou de première ligne) de six mois a été perfectionné dans le années 1970, suite à l'introduction de la rifampicine puis du pyrazinamide. Pendant les 40 ans qui ont suivi, aucun nouveau médicament n'a été développé, ce qui a rendu complexe le traitement des tuberculoses résistantes aux antituberculeux de première ligne. Récemment, deux nouvelles molécules ont été approuvées pour le traitement de la tuberculose M/XDR : la bédaquiline et le delamanid. Toutefois, les essais cliniques qui ont conduit à l'approbation des nouvelles molécules n'ont pas testés ces nouvelles molécules en association, mais en les ajoutant isolément aux régimes les plus classiques. Il est donc primordial d'effectuer des essais cliniques de Phase III pour tester des nouvelles association d'antituberculeux, et en attendant d'effectuer des études observationnelles de bonne qualité méthodologique. Dans le même temps, d'autres antibiotiques, utilisés auparavant pour des infections autre que la tuberculose, ont été détournés pour traiter cette infection ; ce sont les carbapénèmes associés à de l'amoxicilline/clavulanate, la clofazimine, et le linézolide.

  La bédaquiline a été le premier antituberculeux, en dehors des fluoroquinolones, avec un nouveau mécanisme d'action à être approuvée pour le traitement de la tuberculose après plus de 40 ans de pénurie, Il s'agit de la première molécule d'une nouvelle classe d'antituberculeux, les diarylquinolines. La cible de la bédaquiline est l'ATP-synthase, une protéine membranaire qui est fondamentale dans le métabolisme énergétique de la bactérie.Le spectre d'activité de la bédaquiline est très étroit et limité aux mycobactéries. Ce médicament a plusieurs interactions médicamenteuses, en particulier avec les antirétroviraux.La bédaquiline a montré une activité in vitro contre les bacilles « dormants » de M. tuberculosis aussi bien que contre les bacilles en réplication active. Son activité bactéricide est retardée chez l'homme, puisqu'elle apparait après environ 7 jours de traitement. Son activité « stérilisante » semble être synergique avec celle du pyrazinamide. Deux essais cliniques de Phase IIb ont montré que l'addition de la bédaquiline a un traitement antituberculeux théoriquement actif permet d'augmenter le taux de conversion de culture et le taux d'issue de traitement favorable chez des patients atteint de tuberculose à bacilles MDR.Le profil global de tolérance de la bédaquiline apparait plutôt bon ; la bédaquiline a néanmoins été associé à une augmentation de la toxicité hépatique des traitements et une prolongation de l'intervalle QT, mais sans cas d'arythmie cliniquement significative.Plusieurs études observationnelles ont confirmé les résultats des essais cliniques ainsi que de l'intérêt d'utiliser cette molécule pour des durées prolongées de traitement (>24 semaines), voire en association avec le delamanide. Le taux d'apparition de mutants chez M. tuberculosis est comparable à celui de la rifampicine. Une résistance croisée liée à des mutations dans un gène qui code pour des pompes d'efflux a été rapportée entre la bédaquiline et la clofazimine.DelamanideLe delamanide appartient, avec le pretomanide, à la nouvelle classe d'antituberculeux des nitroimidazoles. Les nitroimidazoles inhibent la synthèse de la paroi des mycobactéries ; les mycobactéries représentent en effet la cible principale de cette classe de médicaments. En contraste avec la bédaquiline, le delamanide n'a pas d'interaction médicamenteuse cliniquement significative. L'activité bactéricide précoce du delamanide est faible par rapport aux médicaments de première ligne (isoniazide, rifampicine) ; parmi les nitroimidazoles, une activité stérilisante a jusqu'à présent seulement été démontré pour le pretomanide. Dans un essai clinique de Phase IIa et dans un autre de Phase III, l'addition du delamanide à un traitement théoriquement déjà efficace sur la tuberculose MDR a été associée à un taux plus élevé de négativation des cultures, ainsi qu'à un temps plus court de négativation des cultures. Une association entre l'administration du delamanide et une issue de traitement favorable a été montré dans l'extension observationnelle de l'essai clinique de Phase IIa, mais pas dans l'essai clinique de Phase III. Dans ces essais cliniques, le delamanide a montré une excellente tolérance : les évènements indésirables les plus fréquemment associés au delamanid étaient la prolongation de l'intervalle QT (bien que plus faible que celle causée par la bédaquiline) et une toxicité gastrointestinale. Ces résultats prometteur ont été confirmé par quelques études observationnelles, où le delamanide a été utilisé aussi pour des durées de traitement prolongées et en association avec de la bédaquiline. La résistance au delamanide a été décrite chez quelques cas et semble liée à des mutations dans différents gènes. Il n'y a pas à ce jour de résistance croisée avec d'autres antituberculeux. Travaux originaux Les deux premiers travaux ont porté sur une cohorte rétrospective, observationnelle, et basée sur des données de routine, de cas de tuberculose à bacilles MDR diagnostiqués dans trois centres de référence clinique d'Ile-de-France (Hôpitaux de Bichat, Bligny, et de la Pitié-Salpêtrière) et traités par bédaquiline pendant au moins 30 jours entre 2010 et 2013. Le premier travail avait pour objectif d'évaluer, à six mois de traitement par bédaquiline, à la fois l'efficacité (taux de conversion des cultures et temps de négativation des cultures) et la tolérance de ce nouveau médicament qui était associé à d'autres médicaments comme le linézolide et la clofazimine. Ceci a permis de montrer, dans la plus grosse cohorte mondiale constituée à ce moment-là, que l'efficacité à six mois était très satisfaisante, avec une négativation des cultures des crachats chez tous les malades, même chez des patients ayant une atteinte pulmonaire étendue et un profil de résistance compliqué. De plus, la toxicité était faible et il n'y avait pas eu d'arythmies cardiaques ni de toxicité hépatique grave.

  Dans un troisième travail, nous avons effectué une étude cas-cas au sein d'une cohorte monocentrique des malades atteints par tuberculose pulmonaire MDR et hospitalises dans l'Hôpital de Bligny, Sanatorium de référence au niveau national, entre 2006 et 2014. L'objectif de ce travail était de comparer le taux et le temps de conversion de la culture des crachats à deux et six mois de traitement entre les malades de cette cohorte traités par bédaquiline et des malades traités par fluoroquinolones, la classe d'antibiotiques considérée à ce jour comme la plus efficace pour traiter la tuberculose MDR. Nous avons montré qu'à 6 mois, le taux de conversion de la culture des crachats était plus élevé chez les patients traités par fluoroquinolones par rapport aux patients traités par bédaquiline. Cette différence disparait après ajustement pour les autres variables qui sont associées au temps de conversion des cultures. Les résultats de cette étude étaient donc favorables par rapport à l'efficacité bactériologique de la bédaquiline, et soulignaient pour la première fois que cette dernière pourrait constituer dans le futur un médicament clé pour le traitement de la tuberculose MDR, au même titre que les fluoroquinolones. Le développement de la résistance est un problème majeur qui suit souvent l'introduction des antituberculeux dans la pratique clinique. Dans le quatrième travail, nous avons évalué la fréquence de résistance phénotypique à la bédaquiline chez Mycobacterium tuberculosis en France et nous avons étudié le mécanisme génotypique de la résistance, ainsi que les caractéristiques des patients atteints par tuberculose à bacilles résistants à ce médicament. Toutes les souches de tuberculose MDR reçues entre Janvier 2014 et Décembre 2015 par le Centre de Référence des Mycobactéries ont été testées par un test phénotypique pour détecter la résistance à la bédaquiline ; en cas de résistance phénotypique, le niveau de résistance à différentes concentrations de cet antibiotique a été estimé, ainsi que la présence de mutations dans le gènes connus pour être associés à la résistance à la bédaquiline. Les résultats de ce travail ont mis en évidence un taux inquiétant de résistance à la bédaquiline chez les souches des patients atteints par une tuberculose à bacilles MDR et traités en France. De façon assez surprenante, des souches résistantes ont été identifiées chez deux patients qui n'avaient jamais reçu ce médicament auparavant. Ces données sont en faveur de tester en routine avec un test phénotypique pour la résistance à la bédaquiline tous les cas de tuberculoses à bacilles MDR pour adapter le traitement et empêcher la sélection de d'avantage aux récents changements des recommandations de l'OMS sur le traitement de la tuberculose MDR, qui a révolutionné les précédents groupes des antituberculeux, en réduisant notamment les indications des injectables et en promouvant un traitement complétement par voie orale. Les grandes caractéristiques du traitement de la tuberculose MDR, comme la durée totale de 18 jusqu'à 24 mois, le nombre de molécules actives à utiliser, et la présence d'une phase d'attaque suivi par une phase d'entretien, ont été confirmées par les résultats de ce travail. Malheureusement, le delamanide n'a pas pu être inclus dans l'analyse à cause fu faible nombre d'effectifs traités par cette molécule. microbiologique de la bédaquiline par le biais d'une comparaison avec les plus actifs parmi les médicaments de deuxième ligne, les fluoroquinolones. Notre étude a montré l'absence de différence dans le taux de conversion des cultures des crachats entre patients traités par bédaquiline et par fluoroquinolones, après ajustement par les caractéristiques principales des malades et des souches, en suggérant pour la première fois que la bédaquiline pourrait avoir un rôle comme médicament clé pour le traitement de la tuberculose M/XDR. Le quatrième travail a été une des premières descriptions de la résistance à la bédaquiline au niveau mondial, et la première en France. L'apparition de la résistance à la bédaquiline était inattendue, en particulier en ce qui concerne les deux cas de résistance primaire ; le Centre National de Référence des Mycobactéries a maintenant adopté les tests de résistance à la bédaquiline (phénotypique et génotypique) comme test de routine pour les souches de M. tuberculosis MDR. L'interprétation de la présence des mutations dans le gène Rv0678, ainsi que de l'augmentation des concentrations minimales inhibitrices pour la bédaquiline, reste toutefois compliqué et leur impacte clinique est encore à démontrer. Les études décrites dans ce travail ont plusieurs limitations. D'abord, la collection des données a été effectué rétrospectivement, par la consultation des dossiers médicaux. Deuxièmement, la taille d'échantillon des études est petite. Troisièmement, les études de cohorte n'avaient pas de groupe de contrôle. Quatrièmement, toutes les études étaient observationnelles.

Table 1 .

 1 Drug-drug interactions of bedaquiline and cytochrome P450 inducers/inhibitors.

	Modified from the endTB Clinical and programmatic guide for patient management with new TB drugs,
	version 4.0. 152

Interaction with cytochrome P450 Drugs Effect and recommendation Strong/moderate inducers of cytochrome P450

		Rifamycins (rifampicin,
		rifapentine, rifabutin);	May decrease blood levels of
		Phenytoin; Carbamazepine;	bedaquiline; avoid use with
		Phenobarbital; St. John's	bedaquiline
		Wort (herbal medicine).
		Oral azole antifungals
	Strong/moderate inhibitors of cytochrome P450	(itraconazole, fluconazole); Macrolides except azithromycin (clarithromycin,	May increase blood levels of bedaquiline; avoid use with bedaquiline
		erythromycin).

Table 2 .

 2 Drug-drug interactions of bedaquiline and antiretrovirals.

	Modified from the endTB Clinical and programmatic guide for patient management with new TB drugs,
	version 4.0. 152
	Interaction with cytochrome P450	Drugs	Recommendation
			Substitute nevirapine or
			integrase inhibitor instead of
	Moderate inducer of cytochrome P450	Efavirenz (EFV)	EFV. Allow a five-day washout of EFV if possible.
			If patient is critically ill, no
			washout period is necessary.
			If possible, use an ARV
			regimen with no PI. One
	Strong/moderate inhibitors	Ritonavir-containing	possible solution is to
	of cytochrome P450	protease inhibitors (PIs)	substitute the PI with an
			integrase inhibitor (e.g.
			dolutegravir or raltegravir).

Table 4 .

 4 Efficacy outcomes of the three bedaquiline Phase IIb trials.

	Study	Time of endpoint (weeks)	Arm	mITT population (N)	Result
	C208 Stage 1	8	Bdq Placebo	23 24	TCC: 78 days Cult Conv: 48% TCC: X days Cult Conv: 9%
	C208 Stage 2	24	Bdq Placebo	79 81	TCC: 83 days Cult Conv: 79% TCC/ 125 days Cult Conv: 58%
		120	Bdq Placebo	66 66	Favourable Outcome: 58% Favourable Outcome: 32%
	C209	24	Bdq	233	TCC: 57 days Cult Conv: 79%
		120	Bdq	205	Favourable Outcome: 61%

Table 5 .

 5 Summary of observational studies describing MDR-TB patients treated with bedaquiline.

	First author	Year	Country	N of patients	Type of study, outcome (for cohorts)
	Borisov 197	2017	15 countries	428	Retrospective cohort, final
	Olayanju 198	2018	South Africa	272	Prospective cohort, final
	Udwadia 199	2017	India	20	Retrospective cohort, final
	Kim 200	2018	South Korea	61	Retrospective cohort (including Bdq/Dlm combination), interim
	Ferlazzo 201	2018	3 countries	28	Retrospective cohort (Bdq/Dlm combination), interim
	Skrahina 202	2016	Belarus	197	Retrospective cohort, interim
	Achar 203	2017	4 countries	27	Retrospective cohort (paediatric), interim
	Ndjeka 204	2015	South Africa	91	Retrospective cohort, interim
	Olaru 205	2017	Germany	30	Retrospective cohort, interim
	Maryandishev 206	2017	3 countries	5	Case series (Bdq/Dlm combination)
	Jaspard 207	2017	France	1	Case report (pregnancy)
	Tiberi 208	2013	Italy	2	Case report
	Lachatre 209	2016	France	1	Case report (Bdq/Dlm combination)
	Danckers 210	2014	United States	1	Case report
	Van Halsema 211	2016	United Kingdom	1	Case report
	Aung 212	2017	Myanmar	2	Case report
	Tadolini 213	2016	India	1	Case report (Bdq/Dlm combination)
	Lewis 214	2016	United Kingdom	1	Case report (extended Bdq use)
	Chua 215	2015	Singapore	1	Case report
	Udwadia 216	2014	India	5	Case series
	Somoskovi 217	2015	Switzerland	1	Case report
	Bloemberg 218	2015	Switzerland	1	Case report (Bdq/Dlm combination)
	Hoffmann 219	2016	Switzerland	1	Case report

Table 7 .

 7 Drug-drug interactions of delamanid and cytochrome P450 inducers/inhibitors.

	Drug-drug interactions between delamanid and other drugs that induce/inhibit the cytochrome P450.
	Modified from the endTB Clinical and programmatic guide for patient management with new TB drugs,
	version 4.0. 152
	Interaction with cytochrome P450	Drugs	Effect and recommendation
		Rifamycins (rifampicin,
	Strong inducers of cytochrome P450	rifapentine, rifabutin); Phenytoin; Carbamazepine; Phenobarbital; St. John's	May decrease blood levels of delamanid; if possible, avoid use with delamanid.
		Wort (herbal medicine).
		Oral azole antifungals
		(itraconazole, fluconazole);
	Strong/moderate inhibitors	Macrolide antibiotics except	May increase blood levels of
	of cytochrome P450	azithromycin	delamanid; use with caution.
		(clarithromycin,
		erythromycin).

Table 8 .

 8 Drug-drug interactions of delamanid and antiretrovirals.

	Modified from the endTB Clinical and programmatic guide for patient management with new TB drugs,
	version 4.0. 152
	Interaction with cytochrome P450	Drugs	Recommendation
			May increase blood levels of
			delamanid; use with caution.
			If possible, use an
	Strong/moderate inhibitors of	Ritonavir-containing	antiretroviral regimen with
	cytochrome P450	protease inhibitors (PIs)	no PI. One solution is to
			substitute the PI with an
			integrase inhibitor (e.g.

dolutegravir or raltegravir).

Table 9 .

 9 Efficacy outcomes of delamanid trials. outcomes of the two Phase IIb trials(Trial 204 and Trial 208), one observational follow-up study(Observational Study 116), and one Phase III trial (Trial 213) testing delamanid for the treatment of multidrug-resistant tuberculosis.

	Efficacy Study	Endpoint (weeks)	Arm	mITT population (N)	Result
			Dlm 100 mg	141	Cult Conv: 45%
	Trial 204	8	Dlm 200 mg	136	Cult Conv: 42%
			Placebo	125	Cult Conv: 30%
	Trial 208 and		Dlm ≥6 months	192	Favourable Outcome: 75%
	Observational Study 116	120	No Dlm or Dlm ≤2 months	229	Favourable Outcome: 55%
			Dlm	226	Cult Conv: 58%
		8
			Placebo	101	Cult Conv: 54%
	Trial 213*	24	Dlm Placebo	226 101	TCC: 52 days Cult Conv: 88% TCC: 60 days Cult Conv: 86%
			Dlm	339	Favourable Outcome: 81%
		120
			Placebo	170	Favourable Outcome: 81%

mITT = modified intention-to-treat; Dlm = delamanid; TCC = time to sputum culture conversion; Cult Conv = rate of sputum culture conversion.

Table 10 .

 10 Summary of the characteristics of the observational studies describing MDR-TB treatment with delamanid.

	First author	Year	Country	N of patients	Type of study, outcome (for cohorts)
	Chang 242	2018	Hong Kong	11	Retrospective cohort, mostly final
					Retrospective cohort
	Kuksa 243	2017	Latvia	19	(including Bdq/Dlm
					combination), final
					Retrospective cohort
	Ferlazzo 201	2018	3 countries	28	(Bdq/Dlm combination),
					interim
	Hafkin 244	2016	Multicountry	78	Retrospective cohort, interim
	Hewison 245	2017	7 countries	53	Retrospective cohort (including paediatric), interim
					Retrospective cohort
	Kim 200	2018	South Korea	61	(including Bdq/Dlm
					combination), interim
	Mohr 246	2017	South Africa	103	Retrospective cohort, mostly interim
	Mok 247	2018	South Korea	32	Retrospective cohort, interim
	Tadolini 248	2016	Multicountry	19	Retrospective cohort (paediatric), interim
	Bloemberg 218	2015	Switzerland	1	Case report (Bdq/Dlm combination)
	Esposito 249	2014	Italy	1	Case report (paediatric)
	Hoffmann 219	2016	Switzerland	1	Case report
	Lachatre 209	2016	France	1	Case report (Bdq/Dlm combination)
	Maryandishev 206	2017	3 countries	5	Case series (Bdq/Dlm combination)
	Tadolini 213	2016	India	1	Case report (Bdq/Dlm combination)

Table 11 .

 11 Main safety findings in the delamanid Phase IIb and Phase III trials.

	Source: Skripconoka et al, Gler et al. 67,232,240
	Trial	Trial arm	% pts with AE Total SAE	% pts stopping treatment	Total	% deaths During After the end the trial of the trial
		Dlm 100 mg	91.3	9.9	2.5
	Trial 204	Dlm 200 mg	94.4	12.5	3.8	0.2	0.2	0
		Placebo 92.8	11.2	3.1
	Trial 208 and Observational Study 116	Dlm ≥6 months No Dlm or Dlm ≤2	N.A. N.A.	N.A. N.A.	N.A. N.A.	1.0 8.3.	N.A. N.A.	N.A. N.A.
		months
	Trial 213	Dlm Placebo N.A. N.A.	26.1 27.6	2.3 1.8	4.1 3.5	N.A. N.A.	N.A. N.A.

Pts = patients; AE = adverse event; SAE = serious adverse event; Dlm = delamanid; TB = tuberculosis; N.A. = not available.

Table 12 .

 12 Grouping of anti-tuberculosis drugs according to 2018 World Health Organization recommendations.

  The development and approval of bedaquiline and delamanid was a major breakthrough in anti-tuberculosis drug development, a field that had been stagnant for decades. The slow progress in research and development is a consequence of many factors, including lack of funding, scarce economic interest from pharmaceutical companies, and complacency of national governments. It is, however, undeniable that drug development is a lengthy and costly process, and particularly so in the case of antimycobacterials. The recent and almost concomitant availability of a number of new and re-purposed drugs provides an historical window of opportunity to improve tuberculosis treatment, which may not present itself again for many years.

	IV. Future	perspectives	for	drug/regimen
	development for drug-resistant TB

  days; p=0.005). However, in a multivariate proportional hazard model, controls were not significantly associated with faster culture conversion after adjustment with other co-variates. Variables associated with faster time to culture conversion were the absence of lung cavities (hazard ratio (HR) 6.60, 95%CI 3.21-13.56; p<0.001), having a negative sputum smear at treatment start(HR 4.73, 95% CI 1.01-22.08; p=0.048), and female sex(HR 3.22, 95% CI 1.65-6.30; p=0.001).

de résistance. Cette stratégie a effectivement été appliqué au sein du Centre National de Référence aux Mycobactéries à partir de 2016.Au niveau mondial, l'association de bédaquiline et de delamanide a été utilisée dans un nombre très limité de cas, surtout en raison des craintes de toxicité cardiaque de ces deux médicaments. En effet, chacune des deux molécules peut être responsable d'un allongement de l'intervalle QT et cette association n'a jamais été testée dans un essai clinique. Dans ce cinquième travail, nous nous sommes associés avec les collègues de l'Université de Riga, en Lettonie, pour constituer une série de cas de tuberculose à bacilles MDR traités par l'association des deux nouvelles molécules, données soit simultanément soit de manière séquentielle. L'analyse des 10 dossiers a montré un bon profil de tolérance de l'association, en particulier en ce qui concerne la toxicité cardiaque. Le traitement prolongé par ces deux médicaments était associé à des faibles taux d'allongement de l'intervalle QTcF au-delà de 500 ms et n'était pas associé à des épisodes arythmique, y inclus chez les patients traités par bédaquiline et/ou delamanide pendant plus que 24 semaines. Il faut toutefois tempérer ces bons résultats en se rappelant l'effectif faible des cas analysés. Cette analyse a néanmoins, permis de montrer une efficacité prometteuse de l'association bédaquiline-delamanide qui demande confirmation.En 2016, l'OMS a mandaté un groupe de recherche de l'Université de McGill, au Canada, pour effectuer une revue systématique des données disponibles sur les issues de traitement des patients traités pour une tuberculose à bacilles MDR en conduisant une métaanalyse basée sur des données individuelles de chaque malade. Ces résultats devaient être ensuite présentés au comité en charge de mettre à jour les recommandations de l'OMS pour le traitement de la tuberculose MDR. Nous avons eu la chance d'être associés à cette étude multicentrique (le sixième travail) en reprenant les données des études de cohorte (premier, deuxième, et cinquième travail) et en participant à l'interprétation de ces données. L'objectif principal de cette étude était d'estimer l'association de chaque molécule à une issue du traitement favorable. Les objectifs secondaires étaient au nombre de deux : évaluation de l'impact du nombre de molécules utilisées et de la durée du traitement sur le devenir. Les résultats de cette étude sont, de façon assez surprenante, en contraste avec les acquis précédents sur l'efficacité des molécules de deuxième ligne : en particulier, une utilité très limitée des injectables de deuxième ligne, et ses bénéfices clairs de la bédaquiline et du linézolide sur les taux de succès thérapeutique et sur la mortalité associée. Cette étude a servi

SECOND PART: ORIGINAL

PUBLICATIONS Summary of Article 1

Bedaquiline was approved for the treatment of M/XDR-TB by the stringent regulatory authorities of United States and Europe in 2012 and 2014, respectively. Following this approval, the registration of the drug has startedalthough at a slow pace -in a number of countries. Before registration, access to the drug had however been provided to small number of patients through compassionate use programs. In 2011, France was one of the first countries in the world to introduce bedaquiline via a compassionate use program. When the present study was initiated, the evidence base supporting the use of bedaquiline was extremely limited, as shown in the first part of this manuscript. Furthermore, worries existed about the possible cardiotoxicity of this drug, in particular following the higher mortality rate found in the bedaquiline arm compared to the placebo arm of the C208 Stage 2 trial.

The objective of our study was to perform an interim analysis of microbiological efficacy, defined as time to sputum culture conversion and culture conversion rates at 6 months, which is the standard duration of treatment with bedaquiline, and safety, during the first six months of bedaquiline administration in MDR-TB patients treated in France.

We set up a retrospective cohort where all continuous MDR-TB patients having received at least 30 days of treatment of bedaquiline for compassionate use between January 2010 and July 2013 in three referral TB centres in the Ile-de-France region were included.

Overall, 35 patients were included in the cohort, 19 (54%) of whom had XDR-TB. M. tuberculosis isolates were resistant to a median number of nine drugs (range, 5-12). Among 34 patients (97%) with pulmonary TB involvement, 26 (77%) had bilateral lung disease, 29 (85%) had positive smear sputum microscopy at baseline, and 29 (85%) had at least one lung cavity. In addition, 24 patients (69%) were previously treated for TB, including 17 (49%) having received second-line anti-TB drugs. Hence, a majority of patients had severe TB and a long history of treatment, both risk factors for unfavourable treatment outcome. MDR-TB treatment regimens included bedaquiline plus a median of four (range, 2-5) other drugs, including linezolid in 33 (94%), PAS in 26 (74%), and amikacin in 25 (71%) cases.

Microbiology efficacy was estimated on the 29 patients having culture-positive pulmonary TB at the initiation of treatment with bedaquiline: culture conversion was achieved in 28

VI. Article 2

Long-term outcome and safety of prolonged bedaquiline treatment for multidrug-resistant tuberculosis.

Summary of Article 2

Evidence on the long-term safety of bedaquiline as part of M/XDR-TB treatment regimens outside the carefully selected populations included in clinical trials was lacking. Our interim analysis of a 35-patient cohort revealed promising results at six months of treatment.

Hence, we had to follow all patients to the end of treatment to assess their final outcome.

Meanwhile; some patients received bedaquiline for more than six months mainly because there was no other choice available in the drug armamentarium. Therefore, we seized the opportunity of the cohort to assess the interest and safety of prolonged bedaquiline treatment.

In particular, concerns about the possible toxicity of treatment courses of more than 24 weeks of bedaquiline, which were not investigated in trial setting, have led the World Health Organisation to recommend a standardised 24-week treatment duration for bedaquiline. This one-size-fits-all approach is not in line with a comprehensive and individualised management