Lefamulin in Patients with Community-Acquired Bacterial Pneumonia Caused by Atypical Respiratory Pathogens: Pooled Results from Two Phase 3 Trials

Lefamulin was the first systemic pleuromutilin antibiotic approved for intravenous and oral use in adults with community-acquired bacterial pneumonia based on two phase 3 trials (Lefamulin Evaluation Against Pneumonia [LEAP]-1 and LEAP-2). This pooled analysis evaluated lefamulin efficacy and safety in adults with community-acquired bacterial pneumonia caused by atypical pathogens (Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydia pneumoniae). In LEAP-1, participants received intravenous lefamulin 150 mg every 12 h for 5–7 days or moxifloxacin 400 mg every 24 h for 7 days, with optional intravenous-to-oral switch. In LEAP-2, participants received oral lefamulin 600 mg every 12 h for 5 days or moxifloxacin 400 mg every 24 h for 7 days. Primary outcomes were early clinical response at 96 ± 24 h after first dose and investigator assessment of clinical response at test of cure (5–10 days after last dose). Atypical pathogens were identified in 25.0% (91/364) of lefamulin-treated patients and 25.2% (87/345) of moxifloxacin-treated patients; most were identified by ≥1 standard diagnostic modality (M. pneumoniae 71.2% [52/73]; L. pneumophila 96.9% [63/65]; C. pneumoniae 79.3% [46/58]); the most common standard diagnostic modality was serology. In terms of disease severity, more than 90% of patients had CURB-65 (confusion of new onset, blood urea nitrogen > 19 mg/dL, respiratory rate ≥ 30 breaths/min, blood pressure <90 mm Hg systolic or ≤60 mm Hg diastolic, and age ≥ 65 years) scores of 0–2; approximately 50% of patients had PORT (Pneumonia Outcomes Research Team) risk class of III, and the remaining patients were more likely to have PORT risk class of II or IV versus V. In patients with atypical pathogens, early clinical response (lefamulin 84.4–96.6%; moxifloxacin 90.3–96.8%) and investigator assessment of clinical response at test of cure (lefamulin 74.1–89.7%; moxifloxacin 74.2–97.1%) were high and similar between arms. Treatment-emergent adverse event rates were similar in the lefamulin (34.1% [31/91]) and moxifloxacin (32.2% [28/87]) groups. Limitations to this analysis include its post hoc nature, the small numbers of patients infected with atypical pathogens, the possibility of PCR-based diagnostic methods to identify non-etiologically relevant pathogens, and the possibility that these findings may not be generalizable to all patients. Lefamulin as short-course empiric monotherapy, including 5-day oral therapy, was well tolerated in adults with community-acquired bacterial pneumonia and demonstrated high clinical response rates against atypical pathogens.

. Diagnostic modalities for patients with atypical pathogens detected at baseline* (pooled microITT population [combined treatment groups]); * A patient could have had >1 pathogen identified. Multiple isolates of the same species from the same patient identified by the same testing modality were counted only once. Patients were only counted once for each pathogen based on the unique diagnostic modality or combination of diagnostic modalities by which the pathogen was identified. RT-PCR was performed on OP samples; if RT-PCR was positive for M. pneumoniae, OP samples were used for isolation of M. pneumoniae and for subsequent susceptibility testing. On some occasions, RT-PCR and culture were performed in parallel. Inclusion of L. pneumophila as a baseline pathogen from sputum culture did not require an adequate Gram stain. Culture of C. pneumoniae by the local laboratories was allowed per protocol, but it was not cultured successfully by any of the laboratories. † Includes sputum RT-PCR, serology, and OP swab PCR; sputum RT-PCR, OP swab PCR, and OP swab culture; serology, OP swab PCR, and OP swab culture; and sputum RT-PCR, serology, OP swab PCR, and OP swab culture. ‡ Includes urine UAT, sputum RT-PCR, and serology; and sputum culture, urine UAT, sputum RT-PCR, and serology. CABP, community-acquired bacterial pneumonia; microITT, microbiological intent to treat; n, number of patients with the respective baseline pathogen; OP, oropharyngeal; PCR, polymerase chain reaction; RT-PCR, real-time PCR; UAT, urine antigen testing. Multiple isolates of the same species from the same patient identified by the same testing modality were counted only once. Patients were only counted once for each pathogen based on the unique diagnostic modality or combination of diagnostic modalities by which the pathogen was identified. RT-PCR was performed on OP samples; if RT-PCR was positive for M. pneumoniae, OP samples were used for isolation of M. pneumoniae and for subsequent susceptibility testing. On some occasions, RT-PCR and culture were performed in parallel. Inclusion of L. pneumophila as a baseline pathogen from sputum culture did not require an adequate Gram stain. Culture of C. pneumoniae by the local laboratories was allowed per protocol, but it was not cultured successfully by any of the laboratories. † Includes sputum RT-PCR, serology, and OP swab PCR; sputum RT-PCR, OP swab PCR, and OP swab culture; serology, OP swab PCR, and OP swab culture; and sputum RT-PCR, serology, OP swab PCR, and OP swab culture. ‡ Includes urine UAT, sputum RT-PCR, and serology; and sputum culture, urine UAT, sputum RT-PCR, and serology. CABP, community-acquired bacterial pneumonia; microITT, microbiological intent to treat; n, number of patients with the respective baseline pathogen; OP, oropharyngeal; PCR, polymerase chain reaction; RT-PCR, real-time PCR; UAT, urine antigen testing.  [43]. Defined as baseline presence of ≥2 of the following four criteria: temperature < 36 • C or >38 • C; heart rate >90 bpm; respiratory rate > 20 breaths/min; and WBC < 4000 cells/mm 3 , WBC > 12,000 cells/mm 3 , or immature polymorphonuclear neutrophils > 10%. # Patients received a single dose of short-acting systemic antibacterial medication ≤ 72 h before randomization; randomization was stratified and capped such that ≤25% of the total ITT population met these criteria. ** Defined as normal (CrCl ≥ 90 mL/min), mild (CrCl 60-< 90 mL/min), moderate (CrCl 30-< 60 mL/min), and severe (CrCl < 30 mL/min). † † Medical history terms were defined as follows: hypertension = MedDRA HLT "vascular hypertensive disorders NEC"; asthma/COPD = MedDRA HLT "bronchospasm and obstruction"; diabetes mellitus = MedDRA HLT "diabetes mellitus (incl subtypes)". ‡ ‡ Among the subpopulation of patients with atypical pathogens (M. pneumoniae, L. pneumophila, C. pneumoniae), all patients had ≥1 atypical pathogen at baseline, with the corresponding infections being either mono-or polymicrobial. Within those polymicrobial infections that occurred in patients with atypical pathogens, additional baseline pathogens of S. pneumoniae, H. influenzae, M. catarrhalis, and S. aureus were identified.
Gram stain. Culture of C. pneumoniae by the local laboratories was allowed per protocol, but it was not cultured successfully by any of the laboratories. † Includes sputum RT-PCR, serology, and OP swab PCR; sputum RT-PCR, OP swab PCR, and OP swab culture; serology, OP swab PCR, and OP swab culture; and sputum RT-PCR, serology, OP swab PCR, and OP swab culture. ‡ Includes urine UAT, sputum RT-PCR, and serology; and sputum culture, urine UAT, sputum RT-PCR, and serology. CABP, community-acquired bacterial pneumonia; microITT, microbiological intent to treat; n, number of patients with the respective baseline pathogen; OP, oropharyngeal; PCR, polymerase chain reaction; RT-PCR, real-time PCR; UAT, urine antigen testing.

Safety
Among patients with atypical pathogens at baseline, treatment-emergent adverse events (TEAE) rates were generally similar in the lefamulin (34.1% [31/91]) and moxifloxacin (32.2% [28/87]) groups (Table 2); most were mild or moderate in severity, with 4.5% of patients experiencing severe TEAEs. TEAEs rarely led to study drug discontinuation. All serious TEAEs were unrelated to treatment. Results were consistent with those observed in the overall pooled safety population and when reported by atypical pathogen (Table S2). Among patients with atypical pathogens, TEAE system organ classes that occurred in >5% of patients in the lefamulin group were gastrointestinal disorders; infections and infestations; investigations; and respiratory, thoracic, and mediastinal disorders. The most frequently reported individual TEAEs were diarrhea (lefamulin n = 3 [3.3%]; moxifloxacin n = 2 [2.3%]) and nausea (n = 3 [3.3%]; n = 2 [2.3%]); of these events, most (70%) occurred in patients from the LEAP 2 study who received oral dosing. * Defined as M. pneumoniae, L. pneumophila, and C. pneumoniae. † TEAEs started or worsened during or after first study drug administration (an adverse event with an unknown start date or partial date was categorized as a TEAE); patients with multiple events in a given category were only counted once. ‡ TEAEs that were "Definitely", "Probably", or "Possibly" related to the study drug. If the TEAE relationship was missing, it was treated as "Related". § One patient (aged 70 years; PORT risk class II; moderate renal impairment [creatinine clearance 30 to <60 mL/min] at baseline; history of hypertension and COPD; baseline pathogens Haemophilus influenzae, Haemophilus parainfluenzae, and Mycoplasma pneumoniae) in the lefamulin group had a TEAE leading to death after study day 28; the patient died on study day 271 from acute myeloid leukemia (first reported on study day 269). || Assessed in the intent-to-treat population (lefamulin n = 646; moxifloxacin n = 643); details of deaths have been reported elsewhere [40,41]. Although a patient may have had >1 TEAE, the patient was counted only once within an SOC category and once within a PT category. The same patient may have contributed ≥2 PTs in the same SOC category, but the patient was only counted once toward that SOC category. Adverse events were coded using MedDRA version 20.0 (MedDRA MSSO, Herndon, VA, USA).

Discussion
In patients with CABP due to atypical pathogens, oral and IV lefamulin as a shortcourse empiric monotherapy, including as a 5-day therapy, were well tolerated and associated with high clinical response rates (ECR, IACR success, and microbiological response of success). Efficacy and safety results in patients with atypical pathogens were similar in both populations analyzed (microITT and microITT-2) and when assessed by each atypical pathogen. The results were consistent with those of the overall study population, particularly among patients with atypical pathogens and medical history factors that often complicate disease management and may increase morbidity and mortality, including age ≥65 years or history of smoking, asthma/COPD, or diabetes [44][45][46].
Atypical pathogens are increasingly being recognized as a global public health problem [7,45]; however, testing for atypical pathogens in patients with CABP is not standardized, and widespread differences exist in testing frequency and diagnostic approach [8]. Even standard validated diagnostic assays, such as urine antigen testing for Legionella may not be routinely used [8]. In this post hoc pooled analysis of a subset of patients with CABP caused by atypical pathogens (n = 178), most atypical pathogens were identified by ≥1 standard diagnostic modality, and 45% of patients had polymicrobial pneumonia. However, in clinical practice, the use of multiple diagnostic modalities may not always be feasible (e.g., financial limitations) [8].
Difficulties in accurately identifying CABP-causing pathogens in the real-world setting and the presence of polymicrobial infections in adults with CABP underscore the importance of selecting an appropriate empirical antibiotic that effectively and safely treats both typical and atypical pathogens [47,48]. Evidence suggests that providing empiric antibiotic coverage for atypical pathogens may improve clinical outcomes and reduce economic burden. In a meta-analysis of five randomized controlled trials (n = 2011), the clinical failure rate among hospitalized patients with community-acquired pneumonia was significantly lower in patients who did versus did not receive such coverage (relative risk [95% confidence interval], 0.85 [0.73-0.99]; p = 0.037) [49]. Similarly, results of a multicenter, population-based, retrospective cohort study of 827 hospitalized patients with community-acquired pneumonia showed significant (all p < 0.01) benefits with respect to all-cause mortality in patients with (0.9%) versus without (4.9%) atypical coverage, as well as for mean length of stay (10.2 versus 11.6 days, respectively), total hospital cost (USD 1173 versus USD 1511, respectively), and direct antibiotic cost (USD 426 versus USD 503, respectively) [50]. Lefamulin has previously demonstrated potent in vitro activity against the most common typical and atypical CABP pathogens, including drug-resistant strains [34][35][36]51]. The current post hoc pooled analysis further demonstrates that lefamulin provides efficacy and safety generally similar to that of the respiratory fluoroquinolone, moxifloxacin, in patients with CABP caused by atypical pathogens. This analysis was limited by the relatively low number of LEAP-1 and LEAP-2 patients with CABP caused by atypical respiratory pathogens (approximately 14% of the overall pooled study population), although this observation was generally consistent with previous estimates for atypical pathogens in patients with CABP [6]. A strength of this analysis was the use of a wide variety of diagnostic modalities, including standard detection methods such as serology, culture, and urine antigen testing as well as newer methodologies such as real-time qualitative polymerase chain reaction (RT-PCR), to ensure a sufficient population for analysis. The use of PCR-based diagnostic modalities has the potential to identify pathogens that are not etiologically or clinically relevant to a patient's diagnosis. However, our results indicate that clinical response rates were high and similar between treatment groups regardless of whether the analysis population included (microITT population) or excluded (microITT-2 population) patients with baseline pathogens identified using PCR only. The LEAP-1 and LEAP-2 studies were not powered to detect statistically significant differences regarding atypical pathogens, and the results presented herein should be interpreted as exploratory descriptive analyses. Finally, these findings may not be generalizable to all patients with CABP caused by atypical pathogens, as the enrollment criteria for the LEAP-1 and LEAP-2 trials may have excluded some patients who would typically be seen in clinical practice. Most patients had CURB-65 scores of 0-2, reflective of mild to moderate disease, potentially limiting generalizability of the results to patients with more severe disease. However, approximately two-thirds of the patients had a PORT risk class of III or greater and one-quarter had multilobar pneumonia, suggesting that patients with severe pneumonia may have been reasonably represented by the study population.

Study Design and Participants
In LEAP-1, patients were randomized (1:1) to receive IV lefamulin 150 mg every 12 h (q12h) or IV moxifloxacin 400 mg every 24 h (q24h; with alternating placebo doses to maintain blinding). Patients could switch to oral therapy (lefamulin 600 mg q12h or moxifloxacin 400 mg q24h) after 6 IV doses of study drug (approximately 3 days) if predefined improvement criteria were met. Treatment duration ranged from 5-10 days. In the initial study protocol, many patients received 5 days of lefamulin or 7 days of moxifloxacin, but patients with CABP due to L. pneumophila received 10 days of active treatment. A protocol amendment modified the treatment duration to 7 days for most patients in both groups, including those with CABP due to L. pneumophila. In LEAP-2, patients were randomized (1:1) to receive oral lefamulin 600 mg q12h for 5 days or oral moxifloxacin 400 mg q24h for 7 days (with matching oral placebo to maintain blinding).
Patients were included if they were aged ≥18 years with radiographically diagnosed pneumonia, PORT risk class III-V (LEAP-1, ≥25% PORT risk class IV/V) or II-IV (LEAP-2, ≥50% PORT risk class III/IV), acute onset of ≥3 CABP symptoms, ≥2 vital sign abnormalities, and ≥1 other clinical sign or laboratory finding of CABP. Exclusion criteria included ≥2 days of hospitalization within 90 days before symptom onset, receipt of >1 dose of a short-acting (dosing interval more frequent than q24h) oral or IV antibacterial for CABP within 72 h before randomization, severe immunosuppression, significant hepatic disease, creatinine clearance ≤ 30 mL/min, and being at risk of major cardiac events or dysfunction.
Before study initiation, centers obtained study approval from their respective ethics committees or institutional review boards [42]; all patients provided written informed consent. Trials were compliant with the ethical principles of the Declaration of Helsinki, Good Clinical Practice guidelines, and local laws and regulations.

Microbiological Assessments
Baseline atypical pathogens were identified from specimens collected within 24 h of the first dose of study drug. Diagnostic modalities varied by pathogen, and full details have been published previously [55]. Briefly, M. pneumoniae was identified by serology (≥4-fold increase in M. pneumoniae immunoglobulin [Ig] G serum antibody titer to ≥1:160 between baseline sample and convalescent sample collected at late follow-up visit (30 ± 3 days after first study drug dose) using M. pneumoniae antigen substrate slides and immunofluorescent antibody reagents [MBL Bion, Woburn, MA, USA]), culture from oropharyngeal specimens [56], and RT-PCR positive for the community-acquired respiratory distress syndrome toxin gene (mpn372) in sputum [57,58] or for the repMp1 gene in oropharyngeal specimens [59,60]. L. pneumophila was identified by serology (≥4-fold increase in antibody titer to ≥1:128 by L. pneumophila group 1-6 indirect fluorescent antibody assay [Zeus Scientific, Branchburg, NJ, USA]), rapid urine antigen testing (BinaxNOW ® ; Legionella Urinary Antigen Card Abbott Diagnostics Scarborough, Inc., Scarborough, ME, USA), sputum culture, or RT-PCR positive for the ssrA gene in sputum [57]. C. pneumoniae was identified by serology (≥4-fold increase in IgG serum antibody titer using Chlamydia MIF IgG serologic tests [FOCUS Diagnostics, Cypress, CA, USA] between baseline and convalescent samples) or RT-PCR positive for the argR gene in sputum [57]. Susceptibility testing was performed by broth microdilution according to the Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing [61,62]. Confirmatory identification and susceptibility testing of isolates, urine antigen testing, serology, and RT-PCR were performed by a central laboratory (

Efficacy Assessments
Only patients with baseline atypical pathogens were included in the analyses described herein. Within this patient subgroup, efficacy analyses are presented for the microbiological intent-to-treat (microITT) population (randomized patients with ≥1 baseline CABP-causing pathogen) and the microITT-2 population (randomized patients with ≥1 baseline CABP-causing pathogen detected by a method other than PCR). ECR was assessed at 96 ± 24 h after the first study drug dose. Responders were patients who were alive, showed improvement in ≥2 CABP baseline symptoms, had no worsening of any CABP baseline symptom, and did not receive a nonstudy antibiotic for the treatment of CABP. IACR was assessed at the TOC visit, which was 5-10 days after the last study drug dose. IACR success required resolution or improvement of baseline CABP signs/symptoms such that no additional antibacterial therapy was administered for the current episode of CABP. Microbiological response of success at TOC required either microbiologic eradication (absence of the baseline causative pathogen from repeat cultures obtained between end of treatment [within 2 days after the last study drug dose] and TOC) or presumed eradication (i.e., IACR at TOC was success and culture was not repeated at TOC). TEAEs, defined as any event that started or worsened during or after first study drug dose, were presented for the safety population (all randomized patients who received any amount of study drug) and the microITT population.

Statistical Analyses
For this post hoc pooled analysis, descriptive statistics were generated to characterize patient demographics, baseline clinical characteristics, and efficacy and safety outcomes in the subpopulation of patients with baseline atypical pathogens from LEAP-1 and LEAP-2. These results were interpreted as exploratory descriptive analyses; therefore, no inferential testing was conducted.

Conclusions
In conclusion, lefamulin was well tolerated and led to high clinical response rates in adults with CABP caused by atypical pathogens, including when given as 5-day oral therapy, regardless of complications such as age or comorbidity. This post hoc analysis suggests that lefamulin may provide a new empiric IV and oral monotherapy alternative to fluoroquinolones and macrolides in patients with CABP caused by atypical pathogens.
Supplementary Materials: The following supplementary materials are available online at https://www.mdpi.com/article/10.3390/antibiotics10121489/s1, Table S1: Patient demographic and baseline characteristics by atypical pathogen, Table S2: Overall summary of TEAEs by atypical pathogen. Funding: This research was funded by Nabriva Therapeutics. The APC was funded by Nabriva Therapeutics.
Institutional Review Board Statement: These studies were conducted according to the guidelines of the Declaration of Helsinki, and the studies were approved by Institutional Review Boards/Ethics Committees from each of the 130 study sites, the details of which have been published previously [42].
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.