Levofloxacin activity at increasing doses in a murine model of fluoroquinolone-susceptible and -resistant tuberculosis.

High-dose levofloxacin was explored in a clinical trial against multidrug-resistant tuberculosis and failed to show increased efficacy. In this study, we used a murine model to explore the efficacy of a dose increase in levofloxacin monotherapy beyond the maximum dose evaluated in humans. A total of 120 4-week-old female BALB/c mice were intravenously infected with 106 CFU of Mycobacterium tuberculosis H37Rv wild-type (WT) or isogenic H37Rv mutants harboring GyrA A90V or D94G substitutions; the MICs were 0.25, 4, and 6 µg/mL, respectively. Levofloxacin 250 and 500 mg/kg were given every 12 h (q12h) orally for 4 weeks. Pharmacokinetic parameters were determined after five doses. These two regimens decreased lung bacillary load in mice infected with H37Rv WT but not in mice infected with the A90V and D94G mutants. Levofloxacin 250 mg/kg q12h in mice generated pharmacokinetic parameters equivalent to 1,000 mg/d in humans, whereas 500 mg/kg q12h generated a twofold greater exposure than the highest equivalent dose tested in humans (1,500 mg/d). In our dose-response model, the effective concentration at 50% (EC50) produced an AUC/MIC (AUC0-24h/MIC) ratio of 167.9 ± 27.5, and at EC80 it was 281.2 ± 97.3. Based on this model, high-dose levofloxacin regimens above 1,000 mg/d are not expected to cause a significant increase in bactericidal activity. This study suggests no benefit of high-dose levofloxacin above 1,000 mg/d in the treatment of fluoroquinolone-susceptible or -resistant tuberculosis.

atpE Mutation in Mycobacterium tuberculosis Not Always Predictive of Bedaquiline Treatment Failure.

We report the emergence of an atpE mutation in a clinical Mycobacterium tuberculosis strain. Genotypic and phenotypic bedaquiline susceptibility testing displayed variable results over time and ultimately were not predictive of treatment outcome. This observation highlights the limits of current genotypic and phenotypic methods for detection of bedaquiline resistance.

Nontuberculous Mycobacteria under Scrutiny in the Geneva Area (2015-2020).

BACKGROUND: Nontuberculous mycobacteria (NTM) are increasingly identified in industrialized countries, and their role as pathogens is more frequently recognized. The relative prevalence of NTM strains shows an important geographical variability. Thus, establishing the local relative prevalence of NTM strains is relevant and useful for clinicians. METHODS: Retrospective analysis (2015-2020) of a comprehensive database was conducted including all results of cultures for mycobacteria in a University Hospital (Geneva, Switzerland), covering a population of approximately 500,000 inhabitants. All NTM culture-positive patients were included in the analyses. Patients' characteristics, NTM strains, and time to culture positivity were reported. RESULTS: Among 38,065 samples analyzed during the study period, 411 were culture-positive for NTM, representing 236 strains, and 231 episodes of care which occurred in 222 patients. Patients in whom NTM were identified were predominantly female (55%), with a median age of 62 years, and a low BMI (median: 22.6 kg/m2). The Mycobacterium avium complex (MAC) was the most frequently identified group (37% of strains) followed by Mycobacterium gordonae (25%) and Mycobacterium xenopi (12%) among the slowly growing mycobacteria (SGM), while the Mycobacterium chelonae/abscessus group (11%) were the most frequently identified rapidly growing mycobacteria (RGM). Only 19% of all patients were treated, mostly for pulmonary infections: the MAC was the most frequently treated NTM (n = 19, 43% of cases in patients treated) followed by RGM (n = 15, 34%) and M. xenopi (n = 6, 14%). Among those treated, 23% were immunosuppressed, 12% had pulmonary comorbidities, and 5% systemic comorbidities. Cultures became positive after a median of 41 days (IQR: 23; 68) for SGM and 28 days (14; 35) for RGM. CONCLUSIONS: In Western Switzerland, M. avium and M. gordonae were the most prevalent NTM identified. Positive cultures for NTM led to a specific treatment in 19% of subjects. Patients with a positive culture for NTM were mostly female, with a median age of 62 years, a low BMI, and a low prevalence of immunosuppression or associated severe comorbidities.

Non-tuberculous mycobacterial pulmonary diseases in France: an 8 years nationwide study.

BACKGROUND: The objective of the study was to describe the epidemiology, management and cost of non-tuberculous mycobacteria pulmonary disease (NTM-PD) in France. METHODS: A retrospective analysis was performed using the SNDS ("Système national des données de santé") database over 2010-2017. Patients with NTM-PD were identified based on the ICD10 codes during hospitalizations and/or specific antibiotics treatment regimens. The study population was matched (age, sex and region) to a control group (1:3) without NTM-PD. RESULTS: 5628 patients with NTM-PD (men: 52.9%, mean age = 60.9 years) were identified over the study period and 1433 (25.5%) were treated with antibiotics. The proportion of patients still receiving treatment at 6 and 12 months was 40% and 22%, respectively. The prevalence of NTM-PD was estimated at 5.92 per 100,000 inhabitants and the incidence rate of NTM-PD remained stable over time between 1.025/100,000 in 2010 and 1.096/100,000 in 2017. Patients with NTM-PD had more co-morbidities compared to controls: corticoids (57.3% vs. 33.8%), chronic lower respiratory disease (34.4% vs. 2.7%), other infectious pneumonia (24.4% vs. 1.4%), malnutrition (based on hospitalization with the ICD-10 code reported during a hospital stay as a main or secondary diagnosis) (22.0% vs. 2.0%), history of tuberculosis (14.1% vs. 0.1%), HIV (8.7% vs. 0.2%), lung cancer and lung graft (5.7% vs. 0.4%), cystic fibrosis (3.2% vs. 0.0%), gastro-esophageal reflux disease (2.9% vs. 0.9%) and bone marrow transplant (1.3% vs. 0.0%) (p < 0.0001). The mean Charlson comorbidity index score was 1.6 (vs. 0.2 for controls; p < 0.0001). NTM-PD was independently associated with an increased mortality rate with a hazard ratio of 2.8 (95% CI: 2.53; 3.11). Mortality was lower for patients treated with antibiotics compared to untreated patients (HR = 0.772 (95% CI [0.628; 0.949]). Annual total expenses the year following the infection in a societal perspective were € 24,083 (SD: 29,358) in NTM-PD subjects vs. € 3402 (SD: 8575) in controls (p < 0.0001). Main driver of the total expense for NTM-PD patients was hospital expense (> 50% of the total expense). CONCLUSION: Patients with NTM-PD in France were shown to have many comorbidities, their mortality risk is high and mainly driven by NTM-PD, and their management costly. Only a minority of patients got treated with antibiotics and of those patients treated, many stopped their therapy prematurely. These results underline the high burden associated with NTM-PD and the need for improvement of NTM-PD management in France.

Linezolid-Associated Neurologic Adverse Events in Patients with Multidrug-Resistant Tuberculosis, France.

Linezolid is one of the most effective drugs for treating multidrug-resistant tuberculosis (MDR TB), but adverse effects remain problematic. We evaluated 57 MDR TB patients who had received >1 dose of linezolid during 2011-2016. Overall, patients received 600 mg/day of linezolid for a median of 13 months. In 33 (58%) patients, neurologic or ophthalmologic signs developed, and 18 (32%) had confirmed peripheral neuropathy, which for 78% was irreversible at 12 months after the end of TB treatment despite linezolid withdrawal. Among the 19 patients who underwent ophthalmologic evaluation, 14 patients had optic neuropathy that fully reversed for 2. A total of 16 (33%) of 49 patients had a linezolid trough concentration >2 mg/L, and among these, 14 (88%) experienced adverse effects. No significant association was found between trough concentration and neurologic toxicity. These findings suggest the need to closely monitor patients for neurologic signs and discuss optimal duration of linezolid treatment.

Fully weekly antituberculosis regimen: a proof-of-concept study.

BACKGROUND: The World Health Organization recommends supervising the treatment of tuberculosis. Intermittent regimens have the potential to simplify the supervision and improve compliance. Our objective was to analyse the sterilising activity of once-weekly regimens based on drugs with a long half-life, bedaquiline and rifapentine, in a murine model of tuberculosis. METHODS: 300 Swiss mice were infected intravenously infected with ×10-6 CFU Mycobacterium tuberculosis H37Rv. Mice were treated once weekly with regimens containing: 1) bedaquiline, rifapentine and pyrazinamide (BPZ); 2) BPZ plus moxifloxacin (BPZM); 3) BPZM plus clofazimine (BPZMC); 4) the standard daily regimen of tuberculosis. All regimens were given for 4 or 6 months. Bactericidal and sterilising activity were assessed. RESULTS: After 2 months of treatment, the mean count in lungs was 0.76±0.60 log10 CFU in mice treated with the daily control regimen and negative in all mice treated with once-weekly regimens (p<0.05 compared to the daily control). All mice had negative lung cultures on completion of either 4 or 6 months of treatment, whereas 3 months after 4 and 6 months of treatment, respectively, the relapse rate was 64% and 13% in the standard daily regimen, 5% and 0% in BPZ, 0% and 0% in BPMZ and 0% and 5% in BPMZC (p<0.05 for all once-weekly regimens versus 4-month daily control; p>0.05 for all once-weekly regimens versus 6-month daily control). CONCLUSIONS: BPZ-based once-weekly regimens have higher sterilising activity than the standard daily regimen and could greatly simplify treatment administration and possibly shorten the duration of tuberculosis treatment.

Poor Performance of Rapid Molecular Tests to Define Eligibility for the Shortcourse Multidrug-resistant Tuberculosis Regimen.

The World Health Organization recommends shortcourse regimen (SCR) to treat multidrug resistant tuberculosis for patients with strains susceptible by line-probe assays (LPAs) to second-line drugs. Our retrospective study shows LPAs have suboptimal specificity in predicting eligibility for SCR; a quarter of eligible patients would receive inadequate therapy with SCR.

Outcomes of Bedaquiline Treatment in Patients with Multidrug-Resistant Tuberculosis.

Bedaquiline is recommended by the World Health Organization for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). We pooled data from 5 cohorts of patients treated with bedaquiline in France, Georgia, Armenia, and South Africa and in a multicountry study. The rate of culture conversion to negative at 6 months (by the end of 6 months of treatment) was 78% (95% CI 73.5%-81.9%), and the treatment success rate was 65.8% (95% CI 59.9%-71.3%). Death rate was 11.7% (95% CI 7.0%-19.1%). Up to 91.1% (95% CI 82.2%-95.8%) of the patients experienced >1 adverse event, and 11.2% (95% CI 5.0%-23.2%) experienced a serious adverse event. Lung cavitations were consistently associated with unfavorable outcomes. The use of bedaquiline in MDR and XDR TB treatment regimens appears to be effective and safe across different settings, although the certainty of evidence was assessed as very low.

Clinical, Radiological, and Microbiological Characteristics of Mycobacterium simiae Infection in 97 Patients.

Mycobacterium simiae is a rare species of slow-growing nontuberculous mycobacteria (NTM). From 2002 to 2017, we conducted a retrospective study that included all patients with NTM-positive respiratory samples detected in two university hospitals of the French overseas department of Reunion Island. We recorded the prevalence of M. simiae in this cohort, as well as the clinical, radiological, and microbiological features of patients with at least 1 sample positive for M. simiae In our cohort, 97 patients (15.1%) were positive for M. simiae Twenty-one patients (21.6%) met the American Thoracic Society (ATS) criteria for infection. M. simiae infection was associated with bronchiectasis, micronodular lesions, and weight loss. Antibiotic susceptibility testing was performed for 60 patients, and the isolates were found to have low susceptibility to antibiotics, except for amikacin, fluoroquinolones, and clarithromycin. Treatment failed for 4 of the 8 patients treated for M. simiae infection. Here, we describe a specific cluster corresponding to a large cohort of patients with M. simiae, a rare nontuberculous mycobacterium associated with low pathogenicity and poor susceptibility to antibiotics.

Bedaquiline and Linezolid for Extensively Drug-Resistant Tuberculosis in Pregnant Woman.

A woman with extremely drug-resistant tuberculosis treated with a drug regimen including linezolid and bedaquiline during her last 3 weeks of pregnancy gave birth to a child without abnormalities. No fetal toxicities were noted by 2 years after delivery. This drug combination might be safe during the late third trimester of pregnancy.

Molecular Investigation of Resistance to Second-Line Injectable Drugs in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis in France.

The second-line injectable drugs (SLID, i.e., amikacin, kanamycin, capreomycin) are key drugs for the treatment of multidrug-resistant tuberculosis. Mutations in rrs region 1400, tlyA, and eis promoter are associated with resistance to SLID, to capreomycin, and to kanamycin, respectively. In this study, the sequencing data of SLID resistance-associated genes were compared to the results of phenotypic drug susceptibility testing by the proportion method for the SLID in 206 multidrug-resistant clinical isolates of Mycobacterium tuberculosis collected in France. Among the 153 isolates susceptible to the 3 SLID, 145 showed no mutation, 1 harbored T1404C and G1473A mutations in rrs, and 7 had an eis promoter mutation. Among the 53 strains resistant to at least 1 of the SLID, mutations in rrs accounted for resistance to amikacin, capreomycin, and kanamycin for 81%, 75%, and 44% of the isolates, respectively, while mutations in eis promoter were detected in 44% of the isolates resistant to kanamycin. In contrast, no mutations in tlyA were observed in the isolates resistant to capreomycin. The discrepancies observed between the genotypic (on the primary culture) and phenotypic drug susceptibility testing were explained by (i) resistance to SLID with MICs close to the critical concentration used for routine DST and not detected by phenotypic testing (n = 8, 15% of SLID-resistant strains), (ii) low-frequency heteroresistance not detected by sequencing of drug resistance-associated genes on the primary culture (n = 8, 15% of SLID-resistant strains), and (iii) other resistance mechanisms not yet characterized (n = 7, 13% of SLID-resistant strains).

Selection of Resistance to Clarithromycin in Mycobacterium abscessus Subspecies.

Mycobacterium abscessus is an emerging pathogen against which clarithromycin is the main drug used. Clinical failures are commonly observed and were first attributed to acquired mutations in rrl encoding 23S rRNA but were then attributed to the intrinsic production of the erm(41) 23S RNA methylase. Since strains of M. abscessus were recently distributed into subspecies and erm(41) sequevars, we investigated acquired clarithromycin resistance mechanisms in mutants selected in vitro from four representative strains. Mutants were sequenced for rrl, erm(41), whiB, rpIV, and rplD and studied for seven antibiotic MICs. For mutants obtained from strain M. abscessus subsp. abscessus erm(41) T28 sequevar and strain M. abscessus subsp. bolletii, which are both known to produce effective methylase, rrl was mutated in only 19% (4/21) and 32.5% (13/40) of mutants, respectively, at position 2058 (A2058C, A2058G) or position 2059 (A2059C, A2059G). No mutations were observed in any of the other genes studied, and resistance to other antibiotics (amikacin, cefoxitin, imipenem, tigecycline, linezolid, and ciprofloxacin) was mainly unchanged. For M. abscessus subsp. abscessus erm(41) C28 sequevar and M. abscessus subsp. massiliense, not producing effective methylase, 100% (26/26) and 97.5% (39/40) of mutants had rrl mutations at position 2058 (A2058C, A2058G, A2058T) or position 2059 (A2059C, A2059G). The remaining M. abscessus subsp. massiliense mutant showed an 18-bp repeat insertion in rpIV, encoding the L22 protein. Our results showed that acquisition of clarithromycin resistance is 100% mediated by structural 50S ribosomal subunit mutations for M. abscessus subsp. abscessus erm(41) C28 and M. abscessus subsp. massiliense, whereas it is less common for M. abscessus subsp. abscessus erm(41) T28 sequevar and M. abscessus subsp. bolletii, where other mechanisms may be responsible for failure.

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

Bedaquiline, a recently approved drug for the treatment of multidrug-resistant tuberculosis (MDR-TB), is recommended for a duration of 24 weeks. There are scarce data on patients treated with this drug outside clinical trials.All MDR-TB patients who started treatment from January 1, 2011 to December 31, 2013 and received ≥30 days of bedaquiline were included in a multicentre observational cohort.Among 45 MDR-TB patients, 53% harboured isolates resistant to both fluoroquinolones and second-line injectables, and 38% harboured isolates resistant to one of these drug classes. Median bedaquiline treatment duration was 361 days and 33 patients (73%) received prolonged (>190 days) bedaquiline treatment. Overall, 36 patients (80%) had favourable outcome, five were lost to follow-up, three died, and one failed and acquired bedaquiline resistance. No cases of recurrence were reported. Severe and serious adverse events were recorded in 60% and 18% of patients, respectively. Values of Fridericia-corrected QT interval (QTcF) >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 in outcomes or adverse events rates were observed between patients receiving standard and prolonged bedaquiline treatment.Bedaquiline-containing regimens achieved favourable outcomes in a large proportion of patients. Prolonged bedaquiline treatment was overall well tolerated in this cohort.

Evaluation of the new GenoType NTM-DR kit for the molecular detection of antimicrobial resistance in non-tuberculous mycobacteria.

Objectives: Non-tuberculous mycobacteria (NTM) are emerging pathogens causing difficult-to-treat infections. We tested a new assay (GenoType NTM-DR) that detects natural and acquired resistance mechanisms to macrolides and aminoglycosides in frequently isolated NTM species. Methods: Performance was assessed on 102 isolates including reference strains [16 Mycobacterium avium , 10 Mycobacterium intracellulare , 8 Mycobacterium chimaera , 15 Mycobacterium chelonae and 53 Mycobacterium abscessus (including subsp. abscessus isolates, 18 with a t28 in erm(41) and 10 with a c28, 13 subsp. bolletii isolates and 12 subsp. massiliense isolates)]. Genotypes were determined by PCR sequencing of erm(41) and rrl for clarithromycin resistance and of the 1400-1480 rrs region for aminoglycoside resistance. Phenotypes were determined by MIC microdilution. Results: GenoType NTM-DR yielded results concordant with Sanger sequencing for 100/102 (98%) isolates. The erm(41) genotypic pattern was accurately identified for M. abscessus isolates . Mutations in rrl were detected in 15 isolates (7 M. avium complex, 5 M. abscessus and 3 M. chelonae ) with acquired clarithromycin resistance harbouring rrl mutations (a2057c, a2058g, a2058t or a2059c). Mutations in rrs were detected in five isolates with amikacin resistance harbouring the rrs mutation a1408g. In two isolates, the NTM-DR test revealed an rrl mutation (initial sequencing being WT), which was confirmed by re-sequencing. The test results were concordant with phenotypic susceptibility testing in 96/102 (94.1%) isolates, with four clarithromycin-resistant and two amikacin-resistant isolates not harbouring mutations. Conclusions: The GenoType NTM-DR test is efficient in detecting mutations predictive of antimicrobial resistance in M. avium complex, M. abscessus and M. chelonae.

Are moxifloxacin and levofloxacin equally effective to treat XDR tuberculosis?

Background: Moxifloxacin retains partial activity against some fluoroquinolone-resistant mutants of Mycobacterium tuberculosis . Levofloxacin is presumed to be as active as moxifloxacin against drug-susceptible tuberculosis and to have a better safety profile. Objectives: To compare the in vivo activity of levofloxacin and moxifloxacin against M. tuberculosis strains with various levels of fluoroquinolone resistance. Methods: BALB/c mice were intravenously infected with 10 6 M. tuberculosis H37Rv and three isogenic mutants: GyrA A90V, GyrB E540A and GyrB A543V. Treatment with 50 or 100 mg/kg levofloxacin and 60 or 66 mg/kg moxifloxacin was given orally every 6 h, for 4 weeks. Results: Levofloxacin 50 and 100 mg/kg q6h and moxifloxacin 60 and 66 mg/kg q6h generated AUCs in mice equivalent to those of levofloxacin 750 and 1000 mg/day and moxifloxacin 400 and 800 mg/day, respectively, in humans. Moxifloxacin 60 and 66 mg/kg q6h had bactericidal activity against strain H37Rv (MIC ≤ 0.25 mg/L) and mutants GyrB E540A and GyrB A543V (MIC = 0.5 mg/L). Against mutant GyrA A90V (MIC = 2 mg/L), moxifloxacin 60 mg/kg q6h did not prevent bacillary growth, whereas 66 mg/kg q6h had bacteriostatic activity. Levofloxacin 50 mg/kg q6h had bactericidal activity against H37Rv (MIC ≤ 0.25 mg/L) but not against the mutant strains. Levofloxacin 100 mg/kg q6h had bactericidal activity against H37Rv and mutants GyrB E540A (MIC = 0.5 mg/L) and GyrB A543V (MIC= 1 mg/L) but not against mutant GyrA A90V (MIC = 4 mg/L). Conclusions: All mutations reduced fluoroquinolone activity, even those classified as susceptible according to phenotypic tests. High-dose levofloxacin is less effective than high-dose moxifloxacin against both fluoroquinolone-resistant and -susceptible M. tuberculosis strains in mice.

Preliminary Favorable Outcome for Medically and Surgically Managed Extensively Drug-Resistant Tuberculosis, France, 2009-2014.

We report 20 cases of extensively drug-resistant tuberculosis managed in France. Treatment was individualized and included bedaquiline and linezolid for most patients and surgery in 8 patients. At last follow-up (22 months), 19 patients had achieved conversion from positive to negative on culture testing. These promising results of comprehensive management obtained in a small series deserve confirmation.

Performance of the New Version (v2.0) of the GenoType MTBDRsl Test for Detection of Resistance to Second-Line Drugs in Multidrug-Resistant Mycobacterium tuberculosis Complex Strains.

Detecting resistance to fluoroquinolones (FQ) and second-line injectable drugs (amikacin [AMK], kanamycin [KAN], and capreomycin [CAP]) is crucial given the worldwide increase in the incidence of extensively drug-resistant tuberculosis (XDR-TB). A new version of the GenoType MTBDRsl test (v2.0) has been developed to improve the detection of resistance to FQ (involving gyrA and gyrB mutations) and to second-line injectable drugs (involving rrs and eis promoter mutations) in Mycobacterium tuberculosis A collection of 127 multidrug-resistant (MDR) M. tuberculosis complex strains was tested using the first (v1) and second (v2.0) versions of the MTBDRsl test, as well as DNA sequencing. The specificities in resistance detection of v1 and v2.0 were similar throughout, whereas the levels of sensitivity of v2.0 were superior for FQ (94.8% versus 89.6%) and KAN (90.5% versus 59.5%) but similar for AMK (91.3%) and CAP (83.0%). The sensitivity and specificity of v2.0 were superior to those of v1 for the detection of pre-XDR strains (83.3% versus 75.0% and 88.6% versus 67.1%, respectively), whereas the sensitivity of v2.0 was superior to that of v1 only for the detection of XDR strains (83.0% versus 49.1%). In conclusion, MTBDRsl v2.0 is superior to MTBDRsl v1 and efficiently detects the most common mutations involved in resistance to FQ and aminoglycosides/CAP. However, due to mutations not recognized by v2.0 or to the presence of resistance mechanisms not yet characterized (particularly mechanisms related to monoresistance to aminoglycosides or CAP), the results for wild-type strains obtained with MTBDRsl v2.0 should be confirmed by further DNA sequencing and phenotypic drug susceptibility testing.