Microbiology of cystic fibrosis persons not chronically infected with P. aeruginosa: A quasi-experimental study on two different upper airways' sampling methods.

Background: The upper airways of cystic fibrosis (CF) persons are an evolutionary niche where genetically adapted bacterial strains are selected for lung infection. The microbiological studies conducted up to now on the upper airways are not easily comparable. Methods: Using classical culture methods, we simultaneously studied the microbiological status of upper and lower airways in persons not chronically infected with P. aeruginosa. Each person had a single upper airways sampling and a concomitant lower airways sampling. Lower airways sampling was performed by oropharyngeal swab or sputum collection. Using a quasi-experimental design of study, we evaluated the performance of 2 different upper airways' sampling methods, nasal lavage according to method described by Mainz or nasal lavage with a rhino-set. Pain was measured with appropriate scales. Results: A total of 194 persons were enrolled in this study. Pathogenic flora was found in 128 (6.6%) of 194 upper airways samples and in 164 (84.6%) lower airways samples. A statistically significant difference between the upper airways and the lower airways was found in the isolation of S. aureus and non-fermenter gram negatives. Nasal lavage according to Mainz resulted in the isolation of more non-fermenter gramnegatives than the rhino-set (p < 0.05). No differences were found in the pain caused bythe two methods. Conclusions: In our study population, cultures of the upper airway and lower airway differ in CF persons. In people sampled with nasal lavage according to Mainz more non-fermenter gram negatives were detected than with rhino-set. The two sampling methods were comparable with regard to the caused pain, nasal lavage according to Mainz method being quicker to perform.

Lung clearance index in children with cystic fibrosis previously diagnosed with CRMS/CFSPID: A monocentric prospective experience.

INTRODUCTION: No data are available on the values and role of lung clearance index (LCI) in cystic fibrosis (CF) Screen Positive Inconclusive Diagnosis (CFSPID) progressed to CF diagnosis (CFSPID > CF). This study aimed to assess the value of the LCI in correctly predicting the progression of CFSPID to CF. METHODS: This is a prospective study carried out at the CF Regional Center of Florence, Italy from September 1, 2019. We compared LCI values in children with CF diagnosed for positive newborn screening (NBS), CFSPID or CFSPID > CF for pathological sweat chloride (SC). The Exhalyzer-D (EcoMedics AG, Duernten, Switzerland, software version 3.3.1) was used to conduct the LCI tests, every 6 months on stable children. RESULTS: Forty-two cooperating children were enrolled (mean age at LCI tests: 5.4 years, range: 2.7-8.7): 26 (62%) had CF, 8 (19%) were CFSPID > CF for positive SC, while 8 (19%) kept the CFSPID label at last LCI test. The mean LCI value for patients with CF (7.39; 5.98-10.24) was statistically higher compared to both the mean LCI in the CFSPID > CF (6.62; 5.69-7.58) and in CFSPID (6.56; 5.64-7.21). CONCLUSIONS: Most of asymptomatic CFSPID or progressed to CF have normal LCI. Further data on the longitudinal course of LCI during follow up of CFSPID and on larger cohorts is needed.

Nutritional Status and Circulating Levels of Fat-Soluble Vitamins in Cystic Fibrosis Patients: A Cohort Study and Evaluation of the Effect of CFTR Modulators.

BACKGROUND: Improved therapy in CF has led to an overall improvement in nutritional status. The objectives of our study are: to cross-sectionally assess nutritional status and serum levels of fat-soluble vitamins; to retrospectively evaluate the efficacy of modulators on nutritional status and fat-soluble vitamin levels. METHODS: In patients younger than 2 years of age, we evaluated growth, in patients aged 2-18 years, we assessed BMI z-scores, and in adults, we assessed absolute BMI values. Levels of 25(OH)D, vitamins A, and E were measured. RESULTS: A cross-sectional analysis was conducted on 318 patients, 109 (34.3%) with pancreatic sufficiency. Only three patients were under 2 years old. In 135 patients aged 2-18 years, the median BMI z-score was 0.11, and 5 (3.7%) patients had malnutrition (z-score ≤ 2SD). In 180 adults, the median BMI was 21.8 kg/m2. Overall, 15 (13.7%) males (M) and 18 (25.3%) females (F) were underweight (18 < BMI > 20); 3 (2.7%) M and 5 (7.0%) F had a BMI < 18. Suboptimal 25(OH)D levels were found in patients with pancreatic insufficiency. The prevalence of deficiency of vitamins A and E is low. After one year of treatment with modulators, the increase in BMI was more consistent (M: 1.58 ± 1.25 kg/m2 F: 1.77 ± 1.21 kg/m2) in elexacaftor/tezacaftor/ivacaftor (ETI)-treated patients compared with other modulators, with a significant increase in levels of all fat-soluble vitamins. CONCLUSIONS: Malnutrition is present in a limited number of subjects. The prevalence of subjects with suboptimal 25(OH)D levels is high. ETI showed a beneficial effect on nutritional status and circulating levels of fat-soluble vitamins.

Different management approaches and outcome for infants with an inconclusive diagnosis following newborn screening for cystic fibrosis (CRMS/CFSPID) and Pseudomonas aeruginosa isolation.

INTRODUCTION: Evidence is currently lacking to guide the management of cystic fibrosis (CF) transmembrane conductance regulator-related metabolic syndrome CF screen-positive inconclusive diagnosis (CRMS/CFSPID) with Pseudomonas aeruginosa (Pa)-positive respiratory culture. This study assessed the clinical data, management, and outcomes of an Italian cohort of CRMS/CFSPID infants with Pa isolated from their airways. METHODS: Data of Pa-positive CRMS/CFSPID infants born between January 2011 and August 2018 and followed at five CF Italian centres were retrospectively extracted. Further data were collected until June 2021 to assess outcomes, prevalence of subjects treated with antimicrobials, and treatment type and duration. RESULTS: Forty-three asymptomatic CRMS/CFSPID patients (median age on 30 June 2021, 82 months; interquartile range [IQR], 63-98 months) with at least one positive airway culture for non-mucoid Pa (median age at first isolation, 18.7 months; IQR, 7-25 months) were enrolled. Of them, 24 (55.8%) underwent anti-Pa therapy. Pa clearance occurred in 22 (91.6%) of 24 patients versus spontaneous clearance in 16 of 19 (84.2%) untreated patients (chi-square, 0.5737; p = 0.44878). After a median follow-up of 6.2 years (IQR, 3.0-9.9), 7 (16.3%) were diagnosed with CF after a pathological sweat test (median age, 43 months; IQR, 28-77 months), 3 (7%) developed recurrent pancreatitis or isolated bronchiectasis consistent with CFTR-related disorder, and the CRMS/CFSPID classification remained in 33 (76.7%). CONCLUSIONS: Pa detection frequently occurs in asymptomatic infants with CRMS/CFSPID but tends to clear spontaneously. More studies are needed to determine if Pa isolation can predict evolution.

Impact of Pancreatitis-Associated Protein on Newborn Screening Outcomes and Detection of CFTR-Related Metabolic Syndrome (CRMS)/Cystic Fibrosis Screen Positive, Inconclusive Diagnosis (CFSPID): A Monocentric Prospective Pilot Experience.

Pancreatitis-Associated Protein (PAP)-based Cystic Fibrosis (CF) newborn bloodspot screening (NBS) protocols detect less CFTR-Related Metabolic Syndrome (CRMS)/CF Screen Positive, Inconclusive Diagnosis (CFSPID). We prospectively evaluated the impact of PAP as the second step of the CF NBS protocol, before the CFTR genetic analysis, on NBS outcomes and CRMS/CFSPID detection in the Tuscany region, Italy. In parallel to the usual protocol (IRT/DNA, protocol 1), PAP was analyzed in IRT-positive infants (IRT/PAP/DNA, protocol 2) from 1 June 2020 until 31 May 2022. We defined an infant as NBS positive if PAP was >1.8 µg/L for IRT value 99th percentile-100 µg/L or >0.6 µg/L for IRT value >100 µg/L. To increase the positive predictive value (PPV) of protocol 2, we retrospectively lowered the upper IRT range value from 100 to 90 µg/L (modified protocol 2). We identified 8 CF and 13 CRMS/CFSPID with protocol 1, 5 CF and 5 CRMS/CFSPID with protocol 2 and 8 CF and 5 CRMS/CFSPID with modified protocol 2. With the PAP-based protocols, we observed a reduction of sweat tests, healthy carrier detection and a significant increase in PPV to 15.38%. Further data are needed in order to evaluate the outcomes of CRMS/CFSPID after a long follow-up.

Clinical outcomes of a large cohort of individuals with the F508del/5T;TG12 CFTR genotype.

BACKGROUND: In recent years, patients with cystic fibrosis (CF) conductance regulator (CFTR) variant poly(T) sequences have been increasingly reported with a wide spectrum of clinical severity. We describe the long-term clinical outcomes and progression to a CF diagnosis over time in a large Italian cohort of patients carrying the CFTR F508del/5T;TG12 genotype. METHODS: A retrospective analysis of subjects from 10 CF centres in Italy with the F508del/5T;TG12 genotype was performed. Demographic, clinical, microbiological, and biochemical data, as well as information about the follow-ups and complications of the enroled patients, were collected. RESULTS: A total of 129 subjects (54 females; median age: 15.0 years, range: 0-58 years; 59 older than 18 years) were included. In terms of initial diagnoses, 30 were CF (23.3%), 41 were CFTR-related disorder (CFTR-RD) (31.7%), and 58 were CF transmembrane conductance regulator-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis (CRMS/CFSPID) (45.0%). After a median follow-up of 6.7 years (range 0.2-25 years), 15 patients progressed to CF, bringing the total number of CF diagnoses to 45/129 (34.9%). Most of these patients had mild lung diseases with pancreatic sufficiency and a low prevalence of CF-related complications. CONCLUSIONS: At the end of the study, 34.9% of subjects with the CFTR F508del/5T;TG12 genotype were diagnosed with CF. We suggest including patients with the F508del/5T;TG12 genotype in long-term follow-ups.

Outcomes of early repeat sweat testing in infants with cystic fibrosis transmembrane conductance regulator-related metabolic syndrome/CF screen-positive, inconclusive diagnosis.

BACKGROUND: Reaching early and definitive diagnosis in infants with cystic fibrosis (CF) transmembrane conductance regulator-related metabolic syndrome (CRMS)/CF screen-positive, inconclusive diagnosis (CFSPID) is a priority of all CF newborn screening programs. Currently, sweat testing (ST) is the gold standard for CF diagnosis or exclusion. We assessed outcomes in a cohort of Italian CRMS/CFSPID infants who underwent repeat ST in the 1st year of life. METHODS: This multicentre, prospective study analysed clinical data and outcomes in CRMS/CFSPID infants born between September 1, 2018, and December 31, 2019, and followed until June 30, 2020. All subjects underwent CF transmembrane conductance regulator (CFTR) gene sequencing and the search for CFTR macrodeletions/macroduplications, and repeat ST in the 1st year of life. RESULTS: Fifty subjects (median age at end of follow-up, 16 months [range, 7-21 months]) were enrolled. Forty-one (82%) had the first sweat chloride (SC) in the intermediate range. During follow up, 150 STs were performed (range, 1-7/infant). After a median follow-up of 8.5 months (range, 1-16.2 months), 11 (22%) subjects were definitively diagnosed as follows: CF (n = 2 [4%]) at 2 and 5 months, respectively; healthy carrier (n = 8 [16%]), at a median age of 4 months (range, 2-8 months); and healthy (n = 1 [2%]) at 2 months of age. Inconclusive diagnosis remained in 39 (78%) infants. CONCLUSIONS: Early repeat ST in the 1st year of life can shorten the time to definitive diagnosis in screening positive subjects with initial SC levels in the intermediate range.

A survey of the prevalence, management and outcome of infants with an inconclusive diagnosis following newborn bloodspot screening for cystic fibrosis (CRMS/CFSPID) in six Italian centres.

OBJECTIVE: We evaluated the prevalence, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene profile, clinical data, management and outcome for infants with a CFTR-related metabolic syndrome/CF Screen Positive, Inconclusive Diagnosis (CRMS/CFSPID) designation from six Italian centres. METHODS: All newborn bloodspot screening (NBS) positive infants born from January 2011 to August 2018 with a CF diagnosis or a CRMS/CFSPID designation were enrolled. Data on sweat testing, genetics, clinical course and management were collected. RESULTS: We enrolled 257 CF patientsand 336 infants with a CRMS/CFSPID designation (CF: CRMS/CFSPID ratio of 1:1.30).Blood immuno-reactive trypsinogen (IRT) was significantly lower in CRMS/CFSPID infants and the F508del variant accounted for only 20% of alleles. Children with CRMS/CFSPID showed a milder clinical course, pancreatic sufficiency compared to CF infants. Varied practice across centres was identified regarding sweat testing, chest radiograph (8-100%) and salt supplementation (11-90%). Eighteen (5.3%) CRMS/CFSPID infants converted or were reclassified to diagnosis of CF. Four infants (1.3%) developed a clinical feature consistent with a CFTR-related disorder (1.2%). Twenty-seven were re-classified as healthy carriers (8.0%) and 16 as healthy infants (4.8%). CONCLUSIONS: We have identified considerable variability in the evaluation and management of infants with an inconclusive diagnosis following NBS across six Italian centres. CRMS/CFSPID is more regularly seen in this population compared to countries with higher prevalence of F508del.Conversion to a CF diagnosis was recorded in 18 (5.3%) of CRMS/CFSPID infants and in 16 was as a result of increasing sweat chloride concentration.

CRMS/CFSPID Subjects Carrying D1152H CFTR Variant: Can the Second Variant Be a Predictor of Disease Development?

BACKGROUND: There are no predictive factors of evolution of cystic fibrosis (CF) screen positive inconclusive diagnosis subjects (CFSPIDs). AIM: to define the role of the second CFTR variant as a predictive factor of disease evolution in CFSPIDs carrying the D1152H variant. METHODS: We retrospectively evaluated clinical characteristics and outcome of CFSPIDs carrying the D1152H variant followed at five Italian CF centers. CFSPIDs were divided in two groups: Group A: compound heterozygous for D1152H and a CF-causing variant; Group B: compound heterozygous for D1152H and a: (i) non CF-causing variant, (ii) variant with varying clinical consequences, or (iii) variant with unknown significance. The variants were classified according to CFTR2 mutation database. RESULTS: We enrolled 43 CFSPIDs with at least one D1152H variant: 28 (65.1%) were classified in the group A, and 15 (34.9%) in the Group B. CFSPIDs of group A had the first IRT significantly higher compared to those of group B (p < 0.05) and had a more severe clinical outcome during the follow-up. At the end of the study period, after a mean follow-up of 40.6 months (range 6-91.6), 4 (9.3%) out of 43 CFSPIDs progressed to CFTR-RD or CF. All these subjects were in the group A. CONCLUSIONS: The genetic profile could help predict the risk of disease evolution in CFSPIDs carrying D1152H, revealing the subjects that need a more frequent follow-up.

A New Versatile Water-Soluble Iniferter Platform for the Preparation of Molecularly Imprinted Nanoparticles by Photopolymerisation in Aqueous Media.

The multi-step synthesis of a new water-soluble dithiocarbamate iniferter platform for the preparation of nanoparticles and -gels in aqueous solvents by photoinduced living-radical polymerisation is described herein. The water solubility of the dithiocarbamate iniferter was achieved by incorporating two unprotected glucose units into the iniferter structure by copper(I)-catalysed azide-alkyne cycloaddition ("click chemistry"). Molecularly imprinted nanoparticles (MIPs) specific for 2,4-dichlorophenoxyacetic acid and the corresponding non-imprinted particles (NIPs) were prepared in pure water by using the prepared iniferter as photoinitiator. Radioligand binding tests confirmed a high imprinting factor, and the living character of the iniferter was demonstrated by re-initiating a second photochemical polymerisation on the NIP nanoparticles in water by using ethylene glycol methacrylate phosphate. Our newly synthesised structure is a promising tool for iniferter-mediated photopolymerisations in aqueous media for the preparation of biocompatible nanomaterials with high potential for biomedical applications in a bottom-up fashion.

A disposable evanescent wave fiber optic sensor coated with a molecularly imprinted polymer as a selective fluorescence probe.

We have developed a disposable evanescent wave fiber optic sensor by coating a molecularly imprinted polymer (MIP) containing a fluorescent signaling group on a 4-cm long polystyrene optical waveguide. The MIP is composed of a naphthalimide-based fluorescent monomer, which shows fluorescence enhancement upon binding with carboxyl-containing molecules. The herbicide 2,4-dichlorophenoxyacetic acid and the mycotoxin citrinin were used as model analytes. The coating of the MIP was either performed ex-situ, by dip-coating the fiber with MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization on the fiber. The sensing element was interrogated with a fiber-coupled spectrofluorimeter. The fiber optic sensor detects targets in the low nM range and exhibits specific and selective recognition over structural analogs and non-related carboxyl-containing molecules. This technology can be extended to other carboxyl-containing analytes, and to a broader spectrum of targets using different fluorescent monomers.

Modulation of the microenvironment surrounding the active site of penicillin G acylase immobilized on acrylic carriers improves the enzymatic synthesis of cephalosporins.

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of ß-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2) conversion.

Modulation of imprinting efficiency in nanogels with catalytic activity in the Kemp elimination.

The interactions between the template and the functional monomer are a key to the formation of cavities in the imprinted nanogels with high molecular recognition properties. Nanogels with enzyme-like activity for the Kemp elimination have been synthesized using 4-vinylpyridine as the functional monomer and indole as the template. The weak hydrogen bond interaction in the complex is shown to be able to induce very distinctive features in the cavities of the imprinted nanogels. The percentage of initiator used in the polymerisation, ranging from 1% to 3%, although it does not have a substantial effect on the catalytic rate, reduces considerably the imprinting efficiency. The alteration of the template/monomer ratio is also investigated, and the data show that there is considerable loss of imprinting efficiency. In terms of substrate selectivity, a number of experiments have been performed using 5-Cl-benzisoxazole as substrate analogue, as well as 5-nitro-indole as template analogue for the preparation of a different set of nanogels. All the kinetic data demonstrate that the chemical structure of the template is key to the molecular recognition properties of the imprinted nanogels that are closely tailored and able to differentiate among small structural changes.

Characterization and study of the orientation of immobilized enzymes by tryptic digestion and HPLC-MS: design of an efficient catalyst for the synthesis of cephalosporins.

An innovative approach to determine the orientation of penicillin G acylase (PGA) from Escherichia coli covalently immobilized onto solid supports has been developed. This method is based on tryptic digestion of immobilized PGA followed by HPLC-MS analysis of the released peptides which are supposed to be only those exposed toward the reaction medium and not directly bound to the solid support. To this purpose, PGA was immobilized on Eupergit C (acrylic hydrophobic resin) and glyoxyl-agarose (hydrophilic resin) functionalized with epoxy and aldehyde groups, respectively, both involving the Lys residues of the protein. The peptide maps obtained were analyzed to derive the orientation of immobilized PGA, as the position of the detected Lys gave indication concerning the accessibility of the different areas of the protein. The results indicate that PGA immobilization on both supports involves mainly Lys located near the binding pocket (70%). Some differences in the enzyme orientation on the two supports can be deduced by the presence of different unbound Lys residues in the released peptides, specific to each support (Lys 117alpha for PGA-Eupergit C; Lys 163alpha and Lys 165alpha for PGA-glyoxyl-agarose). These results have been correlated with the data obtained in the kinetically controlled synthesis and indicate that the orientation of PGA on both supports is partially unfavorable, driving the active site near the support surface. This type of orientation of the enzyme enhances the effect of the nature of the support and of the binding chemistry on the catalytic properties. The information obtained indicated the most suitable support and activation strategy to design an immobilized acylase with good synthetic properties for preparative processes. The glyoxyl-Eupergit C support with enhanced porosity synergically combines the mechanical stability and synthetic performances of immobilized PGA and was successfully used in the synthesis of several cephalosporins.