16S rRNA-Based Microbiota Profiling Assists Conventional Culture Analysis of Airway Samples from Pediatric Cystic Fibrosis Patients.

16S-based sequencing provides broader information on the respiratory microbial community than conventional culturing. However, it (often) lacks species- and strain-level information. To overcome this issue, we used 16S rRNA-based sequencing results from 246 nasopharyngeal samples obtained from 20 infants with cystic fibrosis (CF) and 43 healthy infants, which were all 0 to 6 months old, and compared them to both standard (blind) diagnostic culturing and a 16S-sequencing-informed "targeted" reculturing approach. Using routine culturing, we almost uniquely detected Moraxella catarrhalis, Staphylococcus aureus, and Haemophilus influenzae (42%, 38%, and 33% of samples, respectively). Using the targeted reculturing approach, we were able to reculture 47% of the top-5 operational taxonomical units (OTUs) in the sequencing profiles. In total, we identified 60 species from 30 genera with a median of 3 species per sample (range, 1 to 8). We also identified up to 10 species per identified genus. The success of reculturing the top-5 genera present from the sequencing profile depended on the genus. In the case of Corynebacterium being in the top 5, we recultured them in 79% of samples, whereas for Staphylococcus, this value was only 25%. The success of reculturing was also correlated with the relative abundance of those genera in the corresponding sequencing profile. In conclusion, revisiting samples using 16S-based sequencing profiles to guide a targeted culturing approach led to the detection of more potential pathogens per sample than conventional culturing and may therefore be useful in the identification and, consequently, treatment of bacteria considered relevant for the deterioration or exacerbation of disease in patients like those with CF. IMPORTANCE Early and effective treatment of pulmonary infections in cystic fibrosis is vital to prevent chronic lung damage. Although microbial diagnostics and treatment decisions are still based on conventional culture methods, research is gradually focusing more on microbiome and metagenomic-based approaches. This study compared the results of both methods and proposed a way to combine the best of both worlds. Many species can relatively easily be recultured based on the 16S-based sequencing profile, and it provides more in-depth information about the microbial composition of a sample than that obtained through routine (blind) diagnostic culturing. Still, well-known pathogens can be missed by both routine diagnostic culture methods as well as by targeted reculture methods, sometimes even when they are highly abundant, which may be a consequence of either sample storage conditions or antibiotic treatment at the time of sampling.

Treatment Strategies for GLILD in Common Variable Immunodeficiency: A Systematic Review.

Introduction: Besides recurrent infections, a proportion of patients with Common Variable Immunodeficiency Disorders (CVID) may suffer from immune dysregulation such as granulomatous-lymphocytic interstitial lung disease (GLILD). The optimal treatment of this complication is currently unknown. Experienced-based expert opinions have been produced, but a systematic review of published treatment studies is lacking. Goals: To summarize and synthesize the published literature on the efficacy of treatments for GLILD in CVID. Methods: We performed a systematic review using the PRISMA guidelines. Papers describing treatment and outcomes in CVID patients with radiographic and/or histologic evidence of GLILD were included. Treatment regimens and outcomes of treatment were summarized. Results: 6124 papers were identified and 42, reporting information about 233 patients in total, were included for review. These papers described case series or small, uncontrolled studies of monotherapy with glucocorticoids or other immunosuppressants, rituximab monotherapy or rituximab plus azathioprine, abatacept, or hematopoietic stem cell transplantation (HSCT). Treatment response rates varied widely. Cross-study comparisons were complicated because different treatment regimens, follow-up periods, and outcome measures were used. There was a trend towards more frequent GLILD relapses in patients treated with corticosteroid monotherapy when compared to rituximab-containing treatment regimens based on qualitative endpoints. HSCT is a promising alternative to pharmacological treatment of GLILD, because it has the potential to not only contain symptoms, but also to resolve the underlying pathology. However, mortality, especially among immunocompromised patients, is high. Conclusions: We could not draw definitive conclusions regarding optimal pharmacological treatment for GLILD in CVID from the current literature since quantitative, well-controlled evidence was lacking. While HSCT might be considered a treatment option for GLILD in CVID, the risks related to the procedure are high. Our findings highlight the need for further research with uniform, objective and quantifiable endpoints. This should include international registries with standardized data collection including regular pulmonary function tests (with carbon monoxide-diffusion), uniform high-resolution chest CT radiographic scoring, and uniform treatment regimens, to facilitate comparison of treatment outcomes and ultimately randomized clinical trials.

Development of the gut microbiota in early life: The impact of cystic fibrosis and antibiotic treatment.

OBJECTIVES: Patients with Cystic Fibrosis (CF) suffer from pancreatic insufficiency, lipid malabsorption and gastrointestinal complaints, next to progressive pulmonary disease. Altered mucosal homoeostasis due to malfunctioning chloride channels results in an adapted microbial composition of the gastrointestinal and the respiratory tract. Additionally, antibiotic treatment has the potential to distort resident microbial communities dramatically. This study aims to investigate early life development of the gut microbial community composition of children with CF compared to healthy infants and to study the independent effects of antibiotics taking into account other clinical and lifestyle factors. STUDY DESIGN: Faecal samples from 20 infants with CF and 45 healthy infants were collected regularly during the first 18 months of life and microbial composition was determined using 16S rRNA based sequencing. RESULTS: We observed significant differences in the overall microbiota composition between infants with CF and healthy infants (p<0.001). Akkermansia and Anaerostipes were significantly more abundant in control infants, whereas Streptococci and E. coli were significantly more abundant in infants with CF, also after correction for several clinical factors (p<0.05). Antibiotic use in infants with CF was associated with a lower alpha diversity, a reduced abundance of Bifidobacterium and Bacteroides, and a higher abundance of Enterococcus. CONCLUSION: Microbial development of the gut is different in infants with CF compared to healthy infants from the first months of life on, and further deviates over time, in part as a result of antibiotic treatment. The resulting dysbiosis may have significant functional consequences for the microbial ecosystem in CF patients.

Concordance between upper and lower airway microbiota in infants with cystic fibrosis.

Nasopharyngeal and oropharyngeal samples are commonly used to direct therapy for lower respiratory tract infections in non-expectorating infants with cystic fibrosis (CF).We aimed to investigate the concordance between the bacterial community compositions of 25 sets of nasopharyngeal, oropharyngeal and bronchoalveolar lavage (BAL) samples from 17 infants with CF aged ∼5 months (n=13) and ∼12 months (n=12) using conventional culturing and 16S-rRNA sequencing.Clustering analyses demonstrated that BAL microbiota profiles were in general characterised by a mixture of oral and nasopharyngeal bacteria, including commensals like Streptococcus, Neisseria, Veillonella and Rothia spp. and potential pathogens like Staphylococcus aureus, Haemophilus influenzae and Moraxella spp. Within each individual, however, the degree of concordance differed between microbiota of both upper respiratory tract niches and the corresponding BAL.The inconsistent intra-individual concordance between microbiota of the upper and lower respiratory niches suggests that the lungs of infants with CF may have their own microbiome that seems seeded by, but is not identical to, the upper respiratory tract microbiome.

Development of the Nasopharyngeal Microbiota in Infants with Cystic Fibrosis.

RATIONALE: Cystic fibrosis (CF) is characterized by early structural lung disease caused by pulmonary infections. The nasopharynx of infants is a major ecological reservoir of potential respiratory pathogens. OBJECTIVES: To investigate the development of nasopharyngeal microbiota profiles in infants with CF compared with those of healthy control subjects during the first 6 months of life. METHODS: We conducted a prospective cohort study, from the time of diagnosis onward, in which we collected questionnaires and 324 nasopharynx samples from 20 infants with CF and 45 age-matched healthy control subjects. Microbiota profiles were characterized by 16S ribosomal RNA-based sequencing. MEASUREMENTS AND MAIN RESULTS: We observed significant differences in microbial community composition (P < 0.0002 by permutational multivariate analysis of variance) and development between groups. In infants with CF, early Staphylococcus aureus and, to a lesser extent, Corynebacterium spp. and Moraxella spp. dominance were followed by a switch to Streptococcus mitis predominance after 3 months of age. In control subjects, Moraxella spp. enrichment occurred throughout the first 6 months of life. In a multivariate analysis, S. aureus, S. mitis, Corynebacterium accolens, and bacilli were significantly more abundant in infants with CF, whereas Moraxella spp., Corynebacterium pseudodiphtericum and Corynebacterium propinquum and Haemophilus influenzae were significantly more abundant in control subjects, after correction for age, antibiotic use, and respiratory symptoms. Antibiotic use was independently associated with increased colonization of gram-negative bacteria such as Burkholderia spp. and members of the Enterobacteriaceae bacteria family and reduced colonization of potential beneficial commensals. CONCLUSIONS: From diagnosis onward, we observed distinct patterns of nasopharyngeal microbiota development in infants with CF under 6 months of age compared with control subjects and a marked effect of antibiotic therapy leading toward a gram-negative microbial composition.