Theodore G. Liou

Summary: After serious infections, many survivors fully recover while others face persistent symptoms including neurocognitive dysfunction, cardiopulmonary instabilities and disabling fatigue, classified broadly as Post-Acute Infection Syndromes (PAIS). Post-Acute Sequelae of COVID19 (PASC) is one special case. To test whether acute infection initiates PAIS, we analyzed proteins in 173 saliva samples collected during severe respiratory viral infections and tracked patient outcomes for five years. We discovered 44 proteins, all derived from interferon stimulated genes, that respond strongly to acute infection. Rates of post-acute syndromes were similar regardless of infection, but the types of Sequelae differed by virus. High expression of 19 proteins predict low General Sequelae rates across all viruses. High expression of 23 overlapping proteins predict low Neurocognitive Sequelae rates only with SARS-CoV-2 infections. We propose that low expressions of these proteins, which control viral replication, innate immunity, and secondary autoimmunity, combine to precipitate PAIS and PASC. Highlights: Acute viral infections elicit interferon stimulated gene (ISG) protein responses. 23 ISG proteins prevent post-infection General Sequelae for all viral infections. 19 ISG proteins prevent post-infection Neurocognitive Sequelae only with SARS-CoV-2. Poor protein responses induce distinct post-viral syndromes with or without COVID19.

Cystic fibrosis (CF) pulmonary exacerbations shorten survival. Treatment guidelines omit minimal inhibitory concentration (MIC)–based antibiotic dose selection. We tested whether MIC-guided intravenous antibiotic strategies prolong time between exacerbations. We retrospectively studied randomly chosen intravenous antibiotic–treated pulmonary exacerbations from 2015 to 2018, followed through 2021. Using pre-admission sputum culture–derived MICs to select dosing targets, we estimated measured:target ratios for β-lactam steady-state and aminoglycoside area under the curve pharmacokinetics. We used Kaplan-Meier and Cox proportional hazards methods to test whether measured:target ratios were associated with time to next exacerbation. We evaluated serial laboratory measurements with linear mixed-effects models for adverse events. We tested model sensitivities to concurrent treatments and severe acute respiratory syndrome coronavirus 2 pandemic–related events. Compared with measured:target ratios ≤1, β-lactam ratios >1 were associated with a median of 19 more (from 204 to 223) exacerbation-free days (91 exacerbations; hazard ratio [HR], 0.60; 95% CI, 0.37–0.99; P = .045); aminoglycoside ratios >1 were associated with a median of 83 more (from 153 to 236) exacerbation-free days (65 exacerbations; HR, 0.54; 95% CI, 0.31–0.93; P = .026); both antibiotic ratios >1 simultaneously were associated with a median of 210 more (from 185 to 396) exacerbation-free days (60 exacerbations; HR, 0.33; 95% CI, 0.15–0.71; P = .004). No toxic antibiotic levels and few adverse laboratory or clinical events occurred. Models were insensitive to other treatments and pandemic events. MIC-guided antibiotic dosing prolongs time between pulmonary exacerbations with few adverse events. Reduced exacerbation frequency predicts better survival and may improve quality of life for people with CF.

Rationale: Modulator therapies like ivacaftor have revolutionized clinical management of cystic fibrosis, showing marked short-term benefits in trials but heterogeneous findings in long-term observational studies. Since newer modulators have become the standard of care for the majority living with cystic fibrosis in the United States, characterizing long-term effectiveness with real-world data is increasingly difficult because of the lack of contemporary comparator groups for performing between-subjects analyses. Objectives: To determine the extent to which ivacaftor preserves long-term lung function and compare the results of within- and between-subjects analyses for evaluating its real-world effectiveness. Methods: This retrospective cohort study used data from the U.S. Cystic Fibrosis Foundation Patient Registry (2003–2016). We used two approaches to evaluate ivacaftor effectiveness on percent predicted forced expiratory volume in 1 second (ppFEV1): 1) within-subject comparisons of ppFEV1 before and after ivacaftor initiation; and 2) comparisons between ivacaftor-treated and untreated individuals with similar disease pathology. We modeled data from 560 ivacaftor-treated individuals with the G551D variant. For between-subjects comparisons, we used propensity scores to match the treated group with 2,800 untreated F508del homozygous individuals. Modulator initiation bias was assessed and accounted for in each model. Results: Our results showed an initial average improvement in ppFEV1 in ivacaftor-treated children and adults (ranging from 4.54% to 6.53% predicted based on within-subject comparison of before vs. after ivacaftor initiation). There was a slower decline in adults, compared with children. These ivacaftor-treated cohorts experienced less decline relative to their F508del homozygous counterparts (between-group differences in treated vs. control ranged from 0.36% to 0.64% predicted). Both the within- and between-subjects comparisons demonstrated similar degrees of ivacaftor effectiveness. However, small differences between the two approaches were observed in younger individuals. Conclusions: Ivacaftor was associated with improved ppFEV1 across all age groups, with the magnitude of improvement roughly 50% of that observed in clinical trials. The results support the need to account for modulator initiation bias and the use of within-subject analysis in future CFTR (cystic fibrosis transmembrane conductance regulator) modulator effectiveness studies, but caution is advised in younger individuals because of developmental changes that may affect pre- and post-treatment comparability.

Rationale: Evaluating approaches to reduce treatment burden is a research priority among people with cystic fibrosis on highly effective modulators, including elexacaftor-tezacaftor-ivacaftor (ETI). Objectives: We sought to evaluate the impact of discontinuing both hypertonic saline (HS) and dornase alfa (DA) versus continuing both therapies among a subgroup of participants in the SIMPLIFY study who sequentially participated in trials evaluating the independent clinical effects of discontinuing HS and DA. Methods: SIMPLIFY participants ≥12 years old on ETI and constituting a subgroup using both HS and DA at study entry were randomized to the HS or DA trial and then randomized 1:1 to continue or discontinue the applicable therapy for 6 weeks. After completion of the first trial, eligible participants could enroll in the second trial beginning with a 2-week run-in. Study outcomes were compared across the duration of SIMPLIFY participation between a cohort remaining on both therapies during SIMPLIFY and a cohort that sequentially discontinued both as a result of trial randomizations. Multivariable regression models were used to estimate treatment differences, adjusted for time between trials, trial order, baseline age, sex at birth, and percent predicted forced expiratory volume in 1 second (ppFEV1) at study entry. Results: Forty-three participants discontinued both therapies by the end of SIMPLIFY, and 63 remained on both, with overall average ppFEV1 of 96.7% at study entry and 3.9 months as the average duration of follow-up from beginning of the first trial to completion of the second trial, including time between trials. No clinically meaningful difference in the change in ppFEV1 from baseline to completion of the second trial was observed between those who discontinued and those who remained on both therapies (difference: 0.22% off-on; 95% confidence interval = −1.60, 2.03). Changes in lung clearance index at 2.5% starting concentration, patient-reported outcomes, and safety outcomes were also comparable. Patient-reported treatment burden, as measured by a Cystic Fibrosis Questionnaire–Revised subscale, significantly decreased in those who discontinued both therapies. Conclusions: SIMPLIFY participants who sequentially discontinued both HS and DA experienced no meaningful changes in clinical outcomes and reported decreased treatment burden as compared with those who remained on both therapies. These data continue to inform a new era of postmodulator care of people with cystic fibrosis.

Introduction: Patent foramen ovale (PFO) affects about 25% of the population. We studied outcomes in cystic fibrosis (CF). Methods: We conducted a case-control study of patients with CF (PwCF) and age- and sex-matched controls who underwent agitated saline contrast (bubble) echocardiography, 1998–2020. We assessed PFO impacts using linear, logistic, quasi-Poisson, and Cox proportional hazards models. Result: Fifty-nine of 64 PwCF and 88 of 93 controls underwent bubble studies to investigate unexplained hypoxemia or dyspnea. PwCF had higher mean pulmonary artery pressure (PAP: 6.9 mm Hg, 95% confidence interval [CI] = 2.35–11.4), reduced tricuspid annular plane systolic excursion (TAPSE: −3.78 mm, CI = −5.64 to −1.93) and similar right ventricular diastolic sizes. Without hypoxemia, the PFO incidence was similar between PwCF and controls; with hypoxemia, PFO was more common in CF (odds ratio = 5.00, CI = 1.32–19.0). In CF, oxygen supplementation occurred at a percent-predicted forced expiratory volume in 1 s (FEV1%), 22.5 points higher with PFO. Adjusted for FEV1%, PFO was associated with 0.59 more prior-year pulmonary exacerbations (CI = 0.20–0.98) and shorter time to next exacerbation (hazard ratio = 1.86, CI = 1.06–3.26). Associations between PFO and hypoxemia or exacerbations were insensitive to PAP, TAPSE, and CF transmembrane regulator protein modulator treatments. PFO was not associated with CF time to death or lung transplantation (median 1.87 years) adjusted for age, sex, FEV1%, and prior-year exacerbation counts. Conclusion: PFO in CF is associated with hypoxemia at higher FEV1% and more pulmonary exacerbations but not survival.

Background: Cystic fibrosis (CF) is caused by CF transmembrane conductance regulator (CFTR) gene mutations producing dysfunctional CFTR proteins leading to progressive clinical disease. Elexacaftor‐tezacaftor‐ivacaftor (ETI) remarkably improves lung disease but is associated with substantial weight gain.Study Design and Methods: We performed a single‐center longitudinal study predicting 6‐month weight gain after ETI initiation. We used linear mixed effects modeling (LME) to determine association of ETI treatment with changing body mass index (BMI). Using linear regression, we examined BMI prediction models with distinct combinations of main effects to identify a model useful for patient counseling. We used up to eight commonly observed clinical characteristics as input variables (age, sex, percent predicted FEV1 [FEV1%], F508del homozygous state, pancreatic sufficiency, HgbA1c, prior modulator use and prior year number of pulmonary exacerbations).Results: We evaluated 154 patients (19‐73 years old, 54% female, FEV₁% = 19−121, 0−6 prior year pulmonary exacerbations). LME demonstrated an association between ETI use and weight increases. Exhaustive testing suggested a parsimonious linear regression model well‐fitted to data that is potentially useful for counseling. The two variable model shows that on average, BMI decreases by 0.045 (95% Confidence Interval [CI] = −0.069 to −0.021, p < 0.001) for every year of age and increases by 0.322 (CI = 0.142 to 0.502, p = 0.001) for each additional prior year exacerbation at the time of ETI initiation.Interpretation: Young patients with many prior year pulmonary exacerbations likely have the largest 6 month weight gain after starting ETI.

Salmonella enterica serovar Typhimurium strain LT2 is protected by two DNA restriction-modification systems (HsdRMS and Mod-Res) and a Type I bacteriophage exclusion (BREX) system (BrxA-L). The LB5000 strain was constructed to inactivate restriction but not methylation in all three systems and has been available for decades (L. R. Bullas and J. I. Ryu, J Bacteriol 156:471–474, 1983, https://doi.org/10.1128/jb.156.1.471-474.1983 ). However, this strain had been heavily mutagenized and contains hundreds of other mutations, including a few in DNA repair genes. Here, we describe the development of a strain that is only mutated for DNA restriction by the three systems and remains competent for DNA modification. We transferred mutations specific to DNA restriction from LB5000 to a wild-type LT2 background. The hsdR and res mutations affected only restriction in the wild-type background, but the brxC and pglZ mutations for the poorly understood BREX system also reduced modification. Amino acids in an unannotated conserved region of PglX in the BREX system were then randomized. Mutations were identified that specifically affected restriction at 37°C but were found to be temperature sensitive for restriction and methylation when tested at 30°C and 42°C. These mutations in PglX are consistent with a domain that communicates DNA methylation information to other BREX effector proteins. Finally, mutations generated in the specificity domain of PglX may have changed the DNA binding site recognized by the BREX system. IMPORTANCE: The restriction system mutants constructed in this study will be useful for cloning DNA and transferring plasmids from other bacterial species into Salmonella . We verified which mutations in strain LB5000 resulted in loss of restriction for each restriction-modification system and the BREX system by moving these mutations to a wild-type Salmonella background. The methylase PglX was then mutagenized, which adds to our knowledge of the BREX system that is found in many bacteria but is not well understood. These PglX mutations affected restriction and methylation at different temperatures, which suggests that the C-terminal region of PglX may coordinate interactions between the methylase and other BREX system proteins.

Rationale: Studies estimating the rate of lung function decline in cystic fibrosis have been inconsistent regarding the methods used. How the methodology used impacts the validity of the results and comparability between studies is unknown. Objectives: The Cystic Fibrosis Foundation established a work group whose tasks were to examine the impact of differing approaches to estimating the rate of decline in lung function and to provide analysis guidelines. Methods: We used a natural history cohort of 35,252 individuals with cystic fibrosis aged ⩾6 years in the Cystic Fibrosis Foundation Patient Registry (CFFPR), 2003–2016. Modeling strategies using linear and nonlinear forms of marginal and mixed-effects models, which have previously quantified the rate of forced expiratory volume in 1 second (FEV1) decline (percent predicted per year), were evaluated under clinically relevant scenarios of available lung function data. Scenarios varied by sample size (overall CFFPR, medium-sized cohort of 3,000 subjects, and small-sized cohort of 150), data collection/reporting frequency (encounter, quarterly, and annual), inclusion of FEV1 during pulmonary exacerbation, and follow-up length (<2 yr, 2–5 yr, entire duration). Results: Rate of FEV1 decline estimates (percent predicted per year) differed between linear marginal and mixed-effects models; overall cohort estimates (95% confidence interval) were 1.26 (1.24–1.29) and 1.40 (1.38–1.42), respectively. Marginal models consistently estimated less rapid lung function decline than mixed-effects models across scenarios, except for short-term follow-up (both were ∼1.4). Rate of decline estimates from nonlinear models diverged by age 30. Among mixed-effects models, nonlinear and stochastic terms fit best, except for short-term follow-up (<2 yr). Overall CFFPR analysis from a joint longitudinal-survival model implied that an increase in rate of decline of 1% predicted per year in FEV1 was associated with a 1.52-fold (52%) increase in the hazard of death/lung transplant, but the results exhibited immortal cohort bias. Conclusions: Differences were as high as 0.5% predicted per year between rate of decline estimates, but we found estimates were robust to lung function data availability scenarios, except short-term follow-up and older age ranges. Inconsistencies among previous study results may be attributable to inherent differences in study design, inclusion criteria, or covariate adjustment. Results-based decision points reported herein will support researchers in selecting a strategy to model lung function decline most reflective of nuanced, study-specific goals.