Jakko van Ingen

Rationale: Current recommended front-line multidrug therapy (MDT) for Mycobacterium avium complex lung disease (MACLD) produces suboptimal outcomes. Amikacin liposome inhalation suspension (ALIS) with MDT is approved for refractory MACLD; however, clinical trial evidence in newly diagnosed MACLD is lacking. Objectives: To evaluate efficacy and safety of ALIS with MDT in the phase 3b, randomized, double-blind, placebo-controlled superiority trial in adults newly diagnosed with MACLD who have not initiated antibiotics (ENCORE NCT04677569). Methods: Patients (N = 425) were randomized 1:1 to receive ALIS 590 mg (n = 213) or placebo (n = 212) plus azithromycin 250 mg and ethambutol 15 mg/kg once daily for 12 months. Primary endpoint was change from baseline to month 13 in Respiratory Symptom Score (RSS). Pre-specified secondary endpoints included culture conversion (CC) at clinically important timepoints, durable culture conversion (DCC), and safety. Results: Median age was 68.0 years, 62.8% were White, and 79.3% were female. Significantly greater improvements in RSS were observed in the ALIS versus comparator arm at 1 month post-treatment (month 13; LS mean change: 17.77 vs 14.66 points; treatment difference 3.11 points; P=0.0299), with improvements sustained at 3 months post-treatment (month 15; treatment arm difference 4.80 points; nominal P=0.0015). Significantly greater proportions of patients achieved CC by month 13 and DCC at month 15 in the ALIS arm versus comparator arm (82.4% vs 55.6% and 76.2% vs 47.6%, respectively; both P<0.0001). No new safety signals were observed. Conclusions: ALIS with MDT was superior to the comparator arm for improving respiratory symptoms and CC in patients newly diagnosed with MACLD.

Background: “Paradoxical reactions” are a poorly understood phenomenon of clinical worsening during adequate antibiotic tuberculosis treatment. We aimed to characterize and predict paradoxical reactions using clinical, microbiological, and inflammatory factors in human immunodeficiency virus (HIV)–negative patients with pulmonary tuberculosis. Methods: We closely monitored HIV-negative patients aged ≥16 years who were admitted to a Dutch referral center. Paradoxical reactions were defined as new or increased tachypnea with hypoxemia or new or increased need for supplemental oxygen, after exclusion of alternative causes. Blood inflammatory parameters at onset of paradoxical reaction, and 1 and 2 weeks prior were matched for treatment duration to those of patients without paradoxical reactions and compared using Stouffer z scores. Baseline and longitudinal mycobacterial load, clinical parameters, chest radiographic involvement, and antibiotic plasma concentrations were assessed for association with paradoxical reactions. Results: Among 81 HIV-negative patients with pulmonary tuberculosis, 19 (24%) experienced a paradoxical reaction, after a median of 13 days of antituberculosis treatment (interquartile range, 8–18 days). Paradoxical reactions were characterized by higher C-reactive protein level (Stouffer z = 4.22), neutrophil count (z = 3.65), neutrophil-to-lymphocyte ratio (z = 4.94), and ferritin level (z = 4.12) and lower lymphocyte count (z = −3.26), monocyte count (z = −4.72), albumin level (z = −6.33), and hemoglobin level (z = −2.32). More extensive lung involvement and higher mycobacterial loads at initiation of antituberculosis treatment were associated with paradoxical reactions. Antituberculosis drug levels were adequate, and mycobacterial loads declined in both groups. High C-reactive protein and neutrophil-to-lymphocyte ratio and lymphopenia at initiation of antituberculosis treatment predicted paradoxical reactions. Conclusions: Paradoxical reactions in HIV-negative individuals with pulmonary tuberculosis concur with, and are preceded by, outspoken systemic inflammation. Baseline inflammatory parameters may guide future stratified preemptive immunomodulation.

Background: Phage nebulization is an attractive administration strategy for pulmonary infections, including tuberculosis (TB). Yet, it requires penetration into and activity in airway linings and secretions.Methods: We assessed activity of a three-phage combination (Fred313cpm-1Δ33, Muddy_HRM_N00157-2 and Fionnbharth_CG_REM-1) against M. tuberculosis in fresh sputum samples from 35 untreated pulmonary TB patients in Cape Town, South Africa. We also evaluated clinical M. tuberculosis isolates for phage susceptibility and assessed patient serum for presence of phage-specific antibodies and neutralizing activity.Results: Sputum incubation for 24 hours with single phage and phage combination significantly increased time to culture positivity (TTP) compared to the control. For the combination at approximately 108 PFU/mL, the median proportional TTP increase was 4·0% (IQR 0·1–8·2%; p=0·003). Incubation with rifampicin (0·1μg/mL) had no effect. Most clinical isolates were susceptible to all phages (23/34, 68%) and 31/34 (91%) were susceptible to two of the three phages. We found phage-specific IgG in serum samples of a subset of TB patients but no phage-neutralizing effect was observed.Interpretation: Individual phages and the three-phage combination reduced viable mycobacterial load in sputum from pulmonary TB patients. The three-phage combination covered most clinical isolates. No neutralising antibodies were detected. These findings support testing of nebulized phage treatment for pulmonary tuberculosis in clinical trials.

Background: Over 40% of patients with MACLD who receive multidrug therapy (MDT) do not achieve microbiologic success; recurrence is common. Aims and Objectives: We present data from ENCORE (NCT04677569), a phase 3b, randomized, placebo-controlled, multinational, active comparator, superiority trial, which evaluated efficacy and safety of 12 months of amikacin liposome inhalation suspension (ALIS) with MDT in adults newly diagnosed with MACLD who have not initiated antibiotics. Evaluating relapse and new infection rates will further characterize treatment efficacy. Methods: Eligible patients were randomized 1:1 to receive once daily MDT (azithromycin 250 mg and ethambutol 15 mg/kg) with either ALIS 590 mg or comparator (empty liposome control) for 12 months. Recurrence analysis (relapse versus new infection) was conducted using whole genome sequencing and data were reported with descriptive statistics. Results: The ENCORE study met the primary endpoint of improvement in respiratory symptoms in the ALIS arm versus comparator arm at month 13 (P=0.0299). Significantly higher rates of culture conversion (CC) by months 6, 12, and 13, and durable CC at month 15, were observed in the ALIS arm vs comparator arm (P<0.0001). This analysis describes rates of relapse (same MAC species and genotype) or new MAC infection (different MAC species/genotype) among patients with CC. Recurrence was more frequent in the comparator arm versus the ALIS arm, with overall fewer relapses than new infections. No new safety signals were observed. Conclusion: The ENCORE study demonstrated superior respiratory symptom improvement and microbiological efficacy, along with lower recurrence rates in the ALIS arm versus comparator arm in patients with newly diagnosed MACLD who have not initiated antibiotics.

As drug-resistant tuberculosis (DR-TB) cases rise, resistance detection in a timely manner is essential to lead effective treatment and limit transmission. Targeted next-generation sequencing (tNGS) offers quick results with multiple important drugs covered, but assessments regarding its performance for DR-TB diagnostic use compared to whole genome sequencing (WGS) as the most comprehensive genomic-based tool are still limited. This cross-sectional study compared resistance profiles generated by Deeplex Myc-TB tNGS assay with WGS for 116 prospectively-collected rifampicin resistant TB samples from West Java, Indonesia. All 116 samples were subject to paired analysis, the clinical samples were split to be directly processed for tNGS and to be cultivated for culture-based WGS. Both WGS and tNGS were carried out using Illumina MiSeq platform. High concordance of tNGS and WGS were observed across thirteen anti-TB drugs evaluated, particularly for drugs included in the BPaLM regimen. Isoniazid had the lowest concordance of 86.73%. Of 116 samples, 31.03% (n = 36) had discrepant resistance calling from the two methods for one or more drugs, which came from 73 discordant variants identification. The most common source of discrepancy was when tNGS detected a resistance-conferring mutation while WGS did not (54.8%). tNGS could detect mixed infection better than WGS, but WGS was superior in identifying detailed major Mycobacterium tuberculosis lineage of the sample. tNGS showed a good level concordance with WGS in detecting resistance-conferring mutations in rifampicin-resistant TB samples, with a more rapid turnaround time. Continuous update to tNGS panel and mutation catalogue is needed to keep the tool clinically relevant. Importance: Drug-resistant tuberculosis (DR-TB) continues to pose worldwide threat, and newer diagnostic tools to generate quick, comprehensive resistance profile are crucial to provide timely appropriate treatment. Targeted next-generation sequencing (tNGS) is a promising new alternative, but more evidence on its performance is needed to support programmatic adoption. By analysing DR-TB samples with both tNGS and whole genome sequencing (WGS) and evaluating their results’ agreement, this study shows that tNGS works just as well as WGS in detecting TB drug resistance-conferring mutations, confirming its potential for routine diagnostic use. This study also observed that while WGS is superior in identifying Mycobacterium tuberculosis lineage with high resolution, it did not detect mixed infection better than tNGS. Notably, this study demonstrated that tNGS is clinically relevant for DR-TB detection in a high burden setting, providing evidence for programmatic consideration in Indonesia and other settings with similar demographics and TB situation.

Objectives: Azithromycin is a key drug in the treatment of most non-tuberculous mycobacterial (NTM) diseases. Its exposure may be decreased by rifampicin co-administration, but to what extent is largely unknown. We measured azithromycin exposure in an NTM disease patient population and quantified the effect of rifampicin co-administration. Methods: We retrospectively collected plasma azithromycin area-under-the-curve from 0 to 6 hours after administration in mg/L*hours (AUC0-6h), peak (Cmax), and trough (Cmin) concentrations from the TDM service at Radboudumc, The Netherlands. Azithromycin exposure measures were compared between patients with and without concurrent rifampicin use, and within patients who had rifampicin stopped during treatment. Results: We analysed data of 130 patients, of whom 59% had NTM pulmonary disease. The azithromycin geometric mean of AUC0-6h in patients with (n = 48) and without (n = 82) rifampicin were 0.90 versus 1.83 mg/L*h, Cmax 0.22 versus 0.46 mg/L, and Cmin 0.043 versus 0.13 mg/L, respectively. A within-patient comparison in 14 subjects showed geometric means of AUC0-6h, Cmax, and Cmin (90%-CI) with rifampicin were 62% (45%–74%), 58% (38%–72%) and 66% (48%–77%) lower than without rifampicin. Interventions based on TDM enabled a strong increase in exposure to azithromycin. No association between azithromycin exposure and disease outcomes was shown, but the number of patients in these analyses was small. Conclusions: This study provides new population exposure data for TDM of azithromycin in NTM disease. Rifampicin co-administration reduces azithromycin exposure by at least half, underscoring the need for upfront azithromycin dose adjustment, application of TDM, or considering alternative drugs for rifampicin, also considering controversy around its effectiveness and adverse effects.

Phage therapy offers promise to combat antimicrobial resistance, including drug-resistant tuberculosis (TB). Understanding phage activity against Mycobacterium tuberculosis (Mtb) adapted to physiologic microenvironments, such as hypoxia and acidity in granulomas, is essential since these conditions induce non-replicating states. We evaluated a phage combination against Mtb under hypoxic, acidic (pH 5.5), and stationary-phase conditions in vitro . In planktonic Mtb growth conditions, phage concentrations increased around day seven followed by a significant reduction in Mtb H37Rv load, which was maintained over 31 days. Phage addition prevented regrowth was observed with rifampicin and isoniazid alone. Individual phage stability was differentially affected by acidic media conditions, resulting in variability of antimycobacterial activity. In hypoxic conditions and stationary growth experiments, phage titers remained stable over time with no change in mycobacterial load compared to controls. Model-based predictions were able to adequately capture phage-mycobacterial interactions with and without rifampicin. The lack of antimycobacterial activity in assays with non-replicating mycobacteria suggest that phages need actively replicating mycobacteria to exert lytic activity. Stable phage concentrations in assays with non-replicating mycobacteria suggests low grade phage replication in these conditions. Established models can support future study design through simulations of different experimental scenarios.