Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Highly effective cystic fibrosis transmembrane conductance (regulator) modulator therapy: shifting the curve for most while leaving some further behind.

PURPOSE OF REVIEW: Traditional cystic fibrosis (CF) care had been focused on early intervention and symptom mitigation. With the advent of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy (HEMT), in particular, the approval of elexacaftor/tezacaftor/ivacaftor in 2019, there has been a dramatic improvement in outcomes in CF. The purpose of this article is to review the benefits, limitations, and impact of HEMT as well as discuss the new implications, challenges, and hope that modulators bring to people with CF (pwCF). RECENT FINDINGS: HEMT has demonstrated sustained improvement in lung function, nutrition, quality of life, and survival for over 90% of pwCF. As HEMT has delivered such promise, there is a small but significant portion of pwCF who do not benefit from HEMT due to ineligible mutations, intolerance, or lack of accessibility to modulators. SUMMARY: HEMT has significantly improved outcomes, but continued research is needed to understand the new challenges and implications the era of HEMT will bring, as well as how to provide equitable care to those who are unable to benefit from HEMT.

The Inovirus Pf4 Triggers Antiviral Responses and Disrupts the Proliferation of Airway Basal Epithelial Cells.

BACKGROUND: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden. METHODS: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays. RESULTS: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation. CONCLUSIONS: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function.

Delivering a New Future for People With Cystic Fibrosis.

Treatment, prognosis, and quality of life for people with cystic fibrosis (CF) have improved steadily since the initial description of the disease, but most dramatically in the past decade. In 2021, the median predicted survival increased to 53 years, compared with 17 years in 1970. The recent improvement in outcomes is attributable to the advent of cystic fibrosis transmembrane regulator (CFTR) modulators, small molecules that enhance the function of defective CFTR protein. The first CFTR modulator, ivacaftor, received Food and Drug Administration approval in 2011 to treat a single CFTR variant, comprising only 4% of those affected by CF. With the demonstration of efficacy, drug approval has been expanded to other variants. Multiple CFTR modulators used in combination with ivacaftor augment efficacy and increase the number of CFTR variants amenable to therapy. Approval of elexecaftor/tezecaftor/ivacaftor in 2019 increased the number of individuals who could benefit from highly effective modulator therapy (HEMT) to ∼90% of the CF population in the United States. HEMT has been dramatically effective, with overall improvements in lung function, quality of life, nutritional status, and, in women, increased fertility. HEMT may delay the onset of other CF-related comorbidities. Although off-target effects, including hepatotoxicity, drug-drug interactions, and putative mental health issues can complicate use, modulator therapy has been generally well tolerated. Ten percent of people with CF have variants that are not amenable to modulator treatment. HEMT, despite its great cost and limited global access, has brought legitimate hope and changed the lives of a significant majority of individuals and families affected by CF in North America.

Bacteriophage and Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics.

Bacteriophages, viruses that infect and replicate within bacteria, impact bacterial responses to antibiotics in complex ways. Recent studies using lytic bacteriophages to treat bacterial infections (phage therapy) demonstrate that phages can promote susceptibility to chemical antibiotics and that phage/antibiotic synergy is possible. However, both lytic and lysogenic bacteriophages can contribute to antimicrobial resistance. In particular, some phages mediate the horizontal transfer of antibiotic resistance genes between bacteria via transduction and other mechanisms. In addition, chronic infection filamentous phages can promote antimicrobial tolerance, the ability of bacteria to persist in the face of antibiotics. In particular, filamentous phages serve as structural elements in bacterial biofilms and prevent the penetration of antibiotics. Over time, these contributions to antibiotic tolerance favor the selection of resistance clones. Here, we review recent insights into bacteriophage contributions to antibiotic susceptibility, resistance, and tolerance. We discuss the mechanisms involved in these effects and address their impact on bacterial fitness.

Filamentous bacteriophage delays healing of Pseudomonas-infected wounds.

Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.

Biochemical, biophysical, and immunological characterization of respiratory secretions in severe SARS-CoV-2 infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor-stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Oral hymecromone decreases hyaluronan in human study participants.

BACKGROUNDHyaluronan (HA), an extracellular matrix glycosaminoglycan, has been implicated in the pathophysiology of COVID-19 infection, pulmonary hypertension, pulmonary fibrosis, and other diseases, but is not targeted by any approved drugs. We asked whether hymecromone (4-methylumbelliferone [4-MU]), an oral drug approved in Europe for biliary spasm treatment that also inhibits HA in vitro and in animal models, could be repurposed as an inhibitor of HA synthesis in humans.METHODSWe conducted an open-label, single-center, dose-response study of hymecromone in healthy adults. Subjects received hymecromone at 1200 (n = 8), 2400 (n = 9), or 3600 (n = 9) mg/d divided into 3 doses daily, administered orally for 4 days. We assessed safety and tolerability of hymecromone and analyzed HA, 4-MU, and 4-methylumbelliferyl glucuronide (4-MUG; the main metabolite of 4-MU) concentrations in sputum and serum.RESULTSHymecromone was well tolerated up to doses of 3600 mg/d. Both sputum and serum drug concentrations increased in a dose-dependent manner, indicating that higher doses lead to greater exposures. Across all dose arms combined, we observed a significant decrease in sputum HA from baseline after 4 days of treatment. We also observed a decrease in serum HA. Additionally, higher baseline sputum HA levels were associated with a greater decrease in sputum HA.CONCLUSIONAfter 4 days of exposure to oral hymecromone, healthy human subjects experienced a significant reduction in sputum HA levels, indicating this oral therapy may have potential in pulmonary diseases where HA is implicated in pathogenesis.TRIAL REGISTRATIONClinicalTrials.gov NCT02780752.FUNDINGStanford Medicine Catalyst, Stanford SPARK, Stanford Innovative Medicines Accelerator program, NIH training grants 5T32AI052073-14 and T32HL129970.

Biochemical, Biophysical, and Immunological Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We find the percent solids and protein content are greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibit heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observe increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factorâ€"stimulated gene-6 (TSG6) staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicate that increases in HA and DNA in COVID-19 respiratory secretion samples correlate with enhanced inflammatory burden and suggest that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Filamentous Bacteriophages and the Competitive Interaction between Pseudomonas aeruginosa Strains under Antibiotic Treatment: a Modeling Study.

Pseudomonas aeruginosa (Pa) is a major bacterial pathogen responsible for chronic lung infections in cystic fibrosis patients. Recent work has implicated Pf bacteriophages, nonlytic filamentous viruses produced by Pa, in the chronicity and severity of Pa infections. Pf phages act as structural elements in Pa biofilms and sequester aerosolized antibiotics, thereby contributing to antibiotic tolerance. Consistent with a selective advantage in this setting, the prevalence of Pf-positive (Pf+) bacteria increases over time in these patients. However, the production of Pf phages comes at a metabolic cost to bacteria, such that Pf+ strains grow more slowly than Pf-negative (Pf-) strains in vitro. Here, we use a mathematical model to investigate how these competing pressures might influence the relative abundance of Pf+ versus Pf- strains in different settings. Our model suggests that Pf+ strains of Pa cannot outcompete Pf- strains if the benefits of phage production falls onto both Pf+ and Pf- strains for a majority of parameter combinations. Further, phage production leads to a net positive gain in fitness only at antibiotic concentrations slightly above the MIC (i.e., concentrations for which the benefits of antibiotic sequestration outweigh the metabolic cost of phage production) but which are not lethal for Pf+ strains. As a result, our model suggests that frequent administration of intermediate doses of antibiotics with low decay rates and high killing rates favors Pf+ over Pf- strains. These models inform our understanding of the ecology of Pf phages and suggest potential treatment strategies for Pf+ Pa infections. IMPORTANCE Filamentous phages are a frontier in bacterial pathogenesis, but the impact of these phages on bacterial fitness is unclear. In particular, Pf phages produced by Pa promote antibiotic tolerance but are metabolically expensive to produce, suggesting that competing pressures may influence the prevalence of Pf+ versus Pf- strains of Pa in different settings. Our results identify conditions likely to favor Pf+ strains and thus antibiotic tolerance. This study contributes to a better understanding of the unique ecology of filamentous phages in both environmental and clinical settings and may facilitate improved treatment strategies for combating antibiotic tolerance.

Dynamic light scattering microrheology for soft and living materials.

We present a method for using dynamic light scattering in the single-scattering limit to measure the viscoelastic moduli of soft materials. This microrheology technique only requires a small sample volume of 12 µL to measure up to six decades in time of rheological behavior. We demonstrate the use of dynamic light scattering microrheology (DLSµR) on a variety of soft materials, including dilute polymer solutions, covalently-crosslinked polymer gels, and active, biological fluids. In this work, we detail the procedure for applying the technique to new materials and discuss the critical considerations for implementing the technique, including a custom analysis script for analyzing data output. We focus on the advantages of applying DLSµR to biologically relevant materials: breast cancer cells encapsulated in a collagen gel and cystic fibrosis sputum. DLSµR is an easy, efficient, and economical rheological technique that can guide the design of new polymeric materials and facilitate the understanding of the underlying physics governing behavior of naturally derived materials.

Biochemical and Biophysical Characterization of Respiratory Secretions in Severe SARS-CoV-2 (COVID-19) Infections.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19 disease, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e. resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percent solids and protein content are all greatly elevated in COVID-19 compared to heathy control samples and closely resemble levels seen in cystic fibrosis (CF), a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan are major components of respiratory secretions in COVID-19 and are likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. These results highlight the dramatic biophysical properties of COVID-19 respiratory secretions and suggest that DNA and hyaluronan may be viable therapeutic targets in COVID-19 infection.

Area under the curve achievement of once daily tobramycin in children with cystic fibrosis during clinical care.

BACKGROUND: The area under the concentration-time curve over 24 hours (AUC24 ) is frequently utilized to monitor tobramycin exposure in children with cystic fibrosis (CF). An understanding of exposure target achievement during clinical implementation of an AUC24 based approach in children is limited. METHODS: A retrospective chart review was performed in children with CF treated with once daily tobramycin and drug concentration monitoring at a pediatric CF center. During clinical care AUC24 was estimated using a traditional log-linear regression approach (LLR). AUC24 was also estimated retrospectively using a pharmacokinetic model-based Bayesian forecasting approach (BF). AUC24 achievement after both approaches were compared. RESULTS: In 77 treatment courses (mean age, 12.7 ± 5.0 years), a target AUC24 100 to 125 mg h/L was achieved after starting dose in 21 (27%) and after initial dose adjustment in 35 (45%). In the first 7 days of treatment, 24 (32%) required ≥3 dose adjustments, and the mean number of drug concentrations measured was 7.1 ± 3.2. Examination of a BF approach demonstrated adequate prediction of measured tobramycin concentrations (median bias -2.1% [95% CI -3.1 to -1.4]; median precision 7.6% [95% CI, 7.1%-8.2%]). AUC24 estimates utilizing the BF approach were higher than the LLR approach with a mean difference of 6.4 mg h/L (95% CI, 4.8 to 8.0 mg h/L). CONCLUSIONS: Achievement of a narrow AUC24 target is challenging during clinical care, and dose individualization is needed in most children with CF. Implementing a BF approach for estimating AUC24 in children with CF is supported.

Methods for Extraction and Detection of Pf Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis.

Background: There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult. Materials and Methods: In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum. Results: Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies. Conclusion: These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF.

Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections.

Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.

Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis.

Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF.

Cystic fibrosis transmembrane conductance regulator modulators: precision medicine in cystic fibrosis.

PURPOSE OF REVIEW: The aim of this study was to describe the newest development in cystic fibrosis (CF) care, CF transmembrane conductance regulator (CFTR) modulator therapies. RECENT FINDINGS: Phase II results showing CFTR modulator triple therapies are more effective than current CFTR modulators. SUMMARY: CFTR modulator therapy targets the protein defective in CF and boosts its function, but the drug must match mutation pathobiology. Ivacaftor, a CFTR potentiator, was the first modulator approved in 2012, with impressive improvement in lung function and other measures of disease in patients with gating and other residual function mutations (∼10% of CF patients). In 2015, the combination of lumacaftor, a CFTR corrector, and ivacaftor was approved for patients homozygous for the F508del mutation (∼40-50% of the CF population) with positive but less impressive clinical response and 10-20% incidence of intolerance. A next-generation CFTR corrector, tezacaftor, with ivacaftor equally effective and better tolerated than lumacaftor, has also received US Food and Drug Administration approval. Novel CFTR correctors, entering Phase 3 trials in triple modulator combination with tezacaftor-ivacaftor, appear substantially more effective for patients who are homozygous for the F508del mutation and can provide benefit for patients with a single F508del mutation. This offers promise of effective CFTR modulator therapy for nearly 90% of CF patients.

Clinical characteristics and outcomes of pediatric patients with CMV DNA detection in bronchoalveolar lavage fluid.

INTRODUCTION: Cytomegalovirus (CMV) infection can cause severe pulmonary disease in immunocompromised patients. There are no standard diagnostic criteria for CMV pulmonary disease beyond histopathology findings on lung tissue, which is challenging to obtain in pediatric patients. Bronchoalveolar lavage (BAL) fluid is easier to obtain. Since CMV remains latent after primary infection and can potentially reactivate due to any inflammatory response, CMV detection in BAL specimen may not indicate acute CMV pulmonary disease. Thus, we describe the clinical manifestations and outcomes of pediatric patients with CMV detection in BAL fluid. METHODS: We reviewed the clinical, radiologic, and laboratory data of patients <19 years old with a BAL specimen positive for CMV during a 5-year period. RESULTS: Thirty-four encounters in 29 patients were found with CMV detected in their BAL specimen. Half (17/34) of the encounters were in immunocompromised patients. CMV, polymerase chain reaction (PCR) was the most common positive test. Forty-seven percent of the patients had other infections detected in BAL specimens. The majority of patients were never treated for CMV and resolved their acute respiratory illness. Only one patient had probable CMV pulmonary disease. DISCUSSION: CMV is frequently recovered from BAL specimens but does not usually indicate acute CMV pulmonary disease. We would suggest that other diagnoses be considered first, even if CMV is recovered. Pediatr Pulmonol. 2017;52:112-118. © 2016 Wiley Periodicals, Inc.

Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis.

Background. Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are frequently tested for CMV, the clinical utility of such testing remains uncertain. Methods. Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results. Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was performed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the comprehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ≤ .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions. Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis.