Ceftolozane/tazobactam plus tobramycin against free-floating and biofilm bacteria of hypermutable Pseudomonas aeruginosa epidemic strains: Resistance mechanisms and synergistic activity.

OBJECTIVE: Acute exacerbations of biofilm-associated Pseudomonas aeruginosa infections in cystic fibrosis (CF) have limited treatment options. Ceftolozane/tazobactam (alone and with a second antibiotic) has not yet been investigated against hypermutable clinical P. aeruginosa isolates in biofilm growth. This study aimed to evaluate, using an in vitro dynamic biofilm model, ceftolozane/tazobactam alone and in combination with tobramycin at simulated representative lung fluid pharmacokinetics against free-floating (planktonic) and biofilm states of two hypermutable P. aeruginosa epidemic strains (LES-1 and CC274) from adolescents with CF. METHODS: Regimens were intravenous ceftolozane/tazobactam 4.5 g/day continuous infusion, inhaled tobramycin 300 mg 12-hourly, intravenous tobramycin 10 mg/kg 24-hourly, and both ceftolozane/tazobactam-tobramycin combinations. The isolates were susceptible to both antibiotics. Total and less-susceptible free-floating and biofilm bacteria were quantified over 120-168 h. Ceftolozane/tazobactam resistance mechanisms were investigated by whole-genome sequencing. Mechanism-based modelling of bacterial viable counts was performed. RESULTS: Monotherapies of ceftolozane/tazobactam and tobramycin did not sufficiently suppress emergence of less-susceptible subpopulations, although inhaled tobramycin was more effective than intravenous tobramycin. Ceftolozane/tazobactam resistance development was associated with classical (AmpC overexpression plus structural modification) and novel (CpxR mutations) mechanisms depending on the strain. Against both isolates, combination regimens demonstrated synergy and completely suppressed the emergence of ceftolozane/tazobactam and tobramycin less-susceptible free-floating and biofilm bacterial subpopulations. CONCLUSION: Mechanism-based modelling incorporating subpopulation and mechanistic synergy well described the antibacterial effects of all regimens against free-floating and biofilm bacterial states. These findings support further investigation of ceftolozane/tazobactam in combination with tobramycin against biofilm-associated P. aeruginosa infections in adolescents with CF.

Long-term outcomes of necrotizing pneumonia.

INTRODUCTION: Necrotizing pneumonia (NP) is a serious complication of community-acquired pneumonia characterised by the destruction of normal lung parenchyma. No study has evaluated the repercussions of the lung damage in the years following the episode. The aim of this study was to assess the long-term impact on lung function and respiratory symptoms in children hospitalised due to NP. METHODS: We analysed outcomes in children given a diagnosis of NP between January 2003 and April 2016. We selected patients aged more than 4 years capable of undergoing a lung function test, that had been followed up for at least 2 years. The patients completed a respiratory questionnaire and underwent a lung function test. RESULTS: We included a total of 24 patients (12 male). The median age at the time of diagnosis was 28 months, the median length of stay was 15 days, and 18 patients required pleural drainage. The mean duration of follow-up after NP was 8.75 years. During the evaluation, none of the patients exhibited asthma, cough, or exercise-induced symptoms. Three children had a second episode of pneumonia that did not require hospital admission. The spirometry results were the following (given as mean±standard deviation): FEV1 Z-score, -0.47±0.65; FVC Z-score, -0.56±0.73; and FEV1/FVC Z-score, 0.19±0.98. We found no evidence of obstructive pulmonary disease or restrictive patterns. CONCLUSIONS: The long-term outcomes of paediatric NP are good. However, patients exhibited mildly impaired lung function several years after the episode. We recommend follow-up of these patients due to potential impairments in lung function in adulthood.

High flow nasal cannula versus standard low flow nasal oxygen during flexible bronchoscopy in children: A randomized controlled trial.

BACKGROUND: Hypoxemia is the most frequent complication of fiberoptic bronchoscopy (FB) in children. Guidelines recommend oxygen supplementation and conventional nasal prongs (NC) are used for this purpose. The aim of this study was to evaluate if the use of high-flow nasal cannula therapy (HFNC) in children undergoing FB result in a lower incidence of hypoxemia than standard oxygen administration. METHODS: Patients aged 1 month-16 years undergoing elective FB were included in a prospective randomized controlled, nonblinded, single-center clinical trial and randomly assigned to receive oxygen via NC or HFNC. Patients' baseline characteristics were recorded pre-bronchoscopy. The primary outcome was oxygen desaturation during the procedure defined as saturation less than 94%. RESULTS: An intention to treat analysis for 53 patients receiving NC and 51 receiving HFNC, showed HFNC patients were less likely to have hypoxemia than were NC patients (p = .011), with an absolute risk reduction of 0.27 (95% confidence interval [CI]: 0.08-0.45) and a number needed to treat of 3.75 (95% CI: 2.22-12.04). Moderate hypoxemia (SpO2 ≥ 90% and <94%, and <60 s) was observed significantly less often with HFNC than with NC (p = .012). Severe hypoxemia (SpO2 < 90% and >30 s) was not different between groups. Patients undergoing bronchoalveolar lavage (BAL) presented fewer desaturations with HFNC (p = .0003). CONCLUSIONS: HFNC offers optimized oxygenation during elective FB with a significant reduction in desaturations and can be considered for oxygen administration, especially when BAL is performed.

Diagnosis and management of aspiration using fiberoptic endoscopic evaluation of swallowing in a Pediatric Pulmonology Unit.

BACKGROUND AND OBJECTIVES: Swallowing disorders lead to chronic lung aspiration. Early detection and treatment of aspiration in children with dysphagia are important to prevent lung damage. Diagnosis of aspiration, which may be silent, requires an instrumental study such as fiberoptic endoscopic evaluation of swallowing (FEES). Despite its usefulness, it is rarely practiced by pediatric pulmonologists. This study aimed to evaluate the feasibility and utility of FEES performed in the pediatric respiratory unit of a tertiary hospital, analyze the clinical characteristics, endoscopic findings and proposed treatments, and identify the factors associated with penetration or aspiration. METHODS: Medical records of 373 children with suspected aspiration who were referred to the pediatric respiratory unit for FEES were reviewed retrospectively. Clinical characteristics, FEES findings, and the proposed treatments were analyzed. RESULTS: Laryngeal penetration/aspiration was seen in 47.9% of the patients. The most common associated conditions were neurological disease and prematurity. The most frequently observed endoscopic finding was altered laryngeal sensitivity (36.5%). Intervention was recommended in 54.2% of the patients. Complications were not seen during any of the procedures. The multivariate logistic regression model revealed an independent association between aspiration and alterations in laryngeal sensitivity (odds ratio [OR], 5.68), pharyngeal pooling (OR, 11.47), and post-swallowing food residues (OR, 8.08). CONCLUSIONS: The FEES procedure performed by pediatric pulmonologists is a reliable method for diagnosing aspiration in children. It can be safely executed by trained pulmonologists, and significant endoscopic signs other than aspiration can guide in the diagnosis and management recommendations.

Utility of fiberoptic bronchoscopy for difficult airway in neonates.

BACKGROUND: Newborns diagnosed with craniofacial malformations or laryngeal and tracheal alterations may often need advanced airway-management for airway stabilization. Although fiberoptic bronchoscopy (FB) is currently the gold standard for difficult airway management, there is a scarcity of published data on the application of FB in newborns for intubation and controlled extubation (CE). OBJECTIVES: This study describes a case series where FB is used for intubation and/or extubation to manage newborns with difficult airway in either urgent procedures or scheduled ones. METHODS: All FB were carried out on newborns with difficult airway in the neonatal unit over the period January 2005 to December 2018. Patient characteristics were collected from clinical reports, description of the technique from the procedure report. RESULTS: 66 FBs were performed from a total of 40 newborns, a median age of 25 days and a weight of 3217 g. Eighteen were ex-premature babies (45%). Six (15%) had craniocervical malformations. 17 (25.7%) FBs were performed for tracheal intubation (TI), 6 in emergency situations, 34 (51.5%) for CE, and 15 (22.7%) for precise tube placement. Clearing of the airway was achieved in all cases and thus there were no failed TIs. In 32 cases (94.1%), CE was successfully performed. In 6 cases, withdrawal of the ET midprocedure was decided to postpone as 4 of these required extra treatment prior to renewed extubation attempt and 2 needed a tracheostomy. 2 patients required subsequent FB reintubation due to airway pathology. Complications during the procedure were mild desaturations (3%) and deep desaturations (7.5%). CONCLUSIONS: FB is very safe and highly useful when performing intubation in neonates with difficult airway but is essential during extubation to avoid any risk of failure with a potentially fatal outcome. Neonatal units of tertiary hospitals should provide equipment and training to manage these neonates. WHAT IS ALREADY KNOWN?: A failed intubation or extubation can lead to cardiac arrest and anoxic brain damage and/or to death in a neonate. Fiberoptic bronchoscopy is recommended as the safest tool for neonates with difficult airway. However, there is little concrete data published in the current literature to support the recommendations. WHAT IS NEW: Fiberoptic bronchoscopy is an essential tool to avoid the risk of a failed extubation in neonates with difficult airway.

[Long-term outcomes of necrotizing pneumonia]. / Resultados a largo plazo de la neumonía necrotizante.

INTRODUCTION: Necrotizing pneumonia (NP) is a serious complication of community-acquired pneumonia characterised by the destruction of normal lung parenchyma. No study has evaluated the repercussions of the lung damage in the years following the episode. The aim of this study was to assess the long-term impact on lung function and respiratory symptoms in children hospitalised due to NP. METHODS: We analysed outcomes in children given a diagnosis of NP between January 2003 and April 2016. We selected patients aged more than 4 years capable of undergoing a lung function test, that had been followed up for at least 2 years. The patients completed a respiratory questionnaire and underwent a lung function test. RESULTS: We included a total of 24 patients (12 male). The median age at the time of diagnosis was 28 months, the median length of stay was 15 days, and 18 patients required pleural drainage. The mean duration of follow-up after NP was 8.75 years. During the evaluation, none of the patients exhibited asthma, cough, or exercise-induced symptoms. Three children had a second episode of pneumonia that did not require hospital admission. The spirometry results were the following (given as mean ± standard deviation): FEV1 z-score, -0.47±0.65; FVC z-score, -0.56±0.73; and FEV1/FVC z-score, 0.19±0.98. We found no evidence of obstructive pulmonary disease or restrictive patterns. CONCLUSIONS: The long-term outcomes of paediatric NP are good. However, patients exhibited mildly impaired lung function several years after the episode. We recommend follow-up of these patients due to potential impairments in lung function in adulthood.

Recurrent wheezing during the first 3 years of life in a birth cohort of moderate-to-late preterm infants.

BACKGROUND: Data addressing short- and long-term respiratory morbidity in moderate-late preterm infants are limited. We aim to determine the incidence of recurrent wheezing and associated risk and protective factors in these infants during the first 3 years of life. METHODS: Prospective, multicenter birth cohort study of infants born at 32+0 to 35+0  weeks' gestation and followed for 3 years to assess the incidence of physician-diagnosed recurrent wheezing. Allergen sensitization and pulmonary function were also studied. We used multivariate mixed-effects models to identify risk factors associated with recurrent wheezing. RESULTS: A total of 977 preterm infants were enrolled. Rates of recurrent wheezing during year (Y)1 and Y2 were similar (19%) but decreased to 13.3% in Y3. Related hospitalizations significantly declined from 6.3% in Y1 to 0.75% in Y3. Independent risk factors for recurrent wheezing during Y2 and Y3 included the following: day care attendance, acetaminophen use during pregnancy, and need for mechanical ventilation. Atopic dermatitis on Y2 and male sex on Y3 were also independently associated with recurrent wheezing. Palivizumab prophylaxis for RSV during the first year of life decreased the risk or recurrent wheezing on Y3. While there were no differences in rates of allergen sensitization, pulmonary function tests (FEV0.5 ) were significantly lower in children who developed recurrent wheezing. CONCLUSIONS: In moderate-to-late premature infants, respiratory symptoms were associated with lung morbidity persisted during the first 3 years of life and were associated with abnormal pulmonary function tests. Only anti-RSV prophylaxis exerted a protective effect in the development of recurrent wheezing.

Neonatal swallowing assessment using fiberoptic endoscopic evaluation of swallowing (FEES).

OBJECTIVES: Swallowing disorders which lead to aspiration are common in premature infants with a postmenstrual age (PMA) of >36 weeks. Aspiration is often silent and the unique symptom is desaturation during feeding. The aim of this study was 1) to determine the number of prematures with desaturations during feeding due to aspiration, using Fiberoptic Endoscopic Evaluation of Swallowing (FEES); 2) to relate clinical factors and FEES findings to aspiration; and 3) to describe type and efficacy of suggested treatments. METHODS: A retrospective review of 62 ex-premature babies with a median PMA of 40 weeks which underwent FEES due to persistent feeding desaturations. Aspiration was related to other FEES findings and to clinical and demographic data. The efficacy of the treatment was evaluated during the FEES and by comparing recorded desaturations during feeding 7 days before and after FEES. RESULTS: A total of 44 (71%) infants were diagnosed with aspiration and/or penetration. No relation was found to demographic or clinical data. The accumulation of saliva and residues post-swallowing were related to aspiration (P < 0.01). In 77.3% of the infants, use of a thickener seemed to reduce aspiration during FEES and was suggested as a treatment. 13.6% of infants received anti-reflux treatment after FEES and 9.1% required gastrostomy. CONCLUSIONS: Aspiration is very frequent in premature infants who present desaturations during feeding and FEES is a useful method for diagnosing and suggesting treatments.

Antibiotic resistance and population structure of cystic fibrosis Pseudomonas aeruginosa isolates from a Spanish multi-centre study.

The first Spanish multi-centre study on the microbiology of cystic fibrosis (CF) was conducted from 2013 to 2014. The study involved 24 CF units from 17 hospitals, and recruited 341 patients. The aim of this study was to characterise Pseudomonas aeruginosa isolates, 79 of which were recovered from 75 (22%) patients. The study determined the population structure, antibiotic susceptibility profile and genetic background of the strains. Fifty-five percent of the isolates were multi-drug-resistant, and 16% were extensively-drug-resistant. Defective mutS and mutL genes were observed in mutator isolates (15.2%). Considerable genetic diversity was observed by pulsed-field gel electrophoresis (70 patterns) and multi-locus sequence typing (72 sequence types). International epidemic clones were not detected. Fifty-one new and 14 previously described array tube (AT) genotypes were detected by AT technology. This study found a genetically unrelated and highly diverse CF P. aeruginosa population in Spain, not represented by the epidemic clones widely distributed across Europe, with multiple combinations of virulence factors and high antimicrobial resistance rates (except for colistin).

Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone.

Emergence of epidemic clones and antibiotic resistance development compromises the management of Pseudomonas aeruginosa cystic fibrosis (CF) chronic respiratory infections. Whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, WGS mutator genotypes (mutome) and resistome of a widespread clone (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. The coexistence of two divergent CC274 clonal lineages was revealed, but without evident geographical barrier; phylogenetic reconstructions and mutational resistome demonstrated the interpatient transmission of mutators. The extraordinary capacity of P. aeruginosa to develop resistance was evidenced by the emergence of mutations in >100 genes related to antibiotic resistance during the evolution of CC274, catalyzed by mutator phenotypes. While the presence of classical mutational resistance mechanisms was confirmed and correlated with resistance phenotypes, results also showed a major role of unexpected mutations. Among them, PBP3 mutations, shaping up ß-lactam resistance, were noteworthy. A high selective pressure for mexZ mutations was evidenced, but we showed for the first time that high-level aminoglycoside resistance in CF is likely driven by mutations in fusA1/fusA2, coding for elongation factor G. Altogether, our results provide valuable information for understanding the evolution of the mutational resistome of CF P. aeruginosa.

Hypoxia Challenge Testing in Neonates for Fitness to Fly.

BACKGROUND: Preflight hypoxia challenge testing (HCT) in a body plethysmograph has previously been done only on infants >3 months of corrected gestational age (CGA). This study aims to determine the earliest fit-to-fly age by testing neonates <1 week old. METHODS: A prospective observational study was carried out on 3 groups of infants: healthy term infants ≤7 days old, preterm infants (≥34 weeks CGA) 2 to 3 days before discharge, and preterm infants with bronchopulmonary dysplasia (BPD). HCT was conducted using a body plethysmograph with a 15% fraction of inspired oxygen. The oxygen saturation (Spo2) test fail point was <85%. RESULTS: Twenty-four term (mean CGA 40 weeks), 62 preterm (37 weeks), and 23 preterm with BPD (39.5 weeks) infants were tested. One term infant (4.2%) and 12 preterm infants without BPD (19.4%) failed. Sixteen (69.3%) preterm infants with BPD failed (P < .001), with a median drop in Spo2 of 16%. At 39 weeks CGA, neither preterm infants without BPD nor term infants had an Spo2 <85%. However, 7 of 12 term infants with BPD failed the HCT. CONCLUSIONS: Term and preterm infants without BPD born at >39 weeks CGA do not appear to be likely to desaturate during a preflight HCT and so can be deemed fit to fly according to current British Thoracic Society Guidelines.

Risk factors for bronchiolitis, recurrent wheezing, and related hospitalization in preterm infants during the first year of life.

BACKGROUND: Airway diseases are highly prevalent in infants and cause significant morbidity. We aimed to determine the incidence and risk factors for respiratory morbidity in a Spanish cohort of moderate-to-late preterm (MLP) infants prospectively followed during their first year of life. METHODS: SAREPREM is a multicenter, prospective, longitudinal study. Preterm infants born at 32-35 weeks of gestation with no comorbidities were enrolled within 2 weeks of life and followed at 2-4 weeks, 6, and 12 months of age. Multivariate mixed-models were performed to identify independent risk factors associated with (i) development of bronchiolitis, (ii) recurrent wheezing, or (iii) related hospital admissions. RESULTS: Overall, 977 preterm infants were included, and 766 (78.4%) completed follow-up. Of those, 365 (47.7%) developed bronchiolitis during the first year, 144 (18.8%) recurrent wheezing, and 48 (6.3%) were hospitalized. While low birthweight, day care attendance (DCA) and school-age siblings were significantly and independently associated with both the development of bronchiolitis and recurrent wheezing, lower maternal age increased the risk for bronchiolitis and respiratory-related hospitalizations. Lastly, mechanical ventilation was associated with a higher risk of bronchiolitis and history of asthma in any parent increased the likelihood of developing recurrent wheezing. CONCLUSIONS: In this study, several non-modifiable parameters (family history of asthma, low birthweight, need for mechanical ventilation) and modifiable parameters (young maternal age, DCA, or exposure to school-age siblings) were identified as significant risk factors for the development of bronchiolitis and recurrent wheezing during the first year of life in MLP infants.

Assessing the improvements in the newborn screening strategy for cystic fibrosis in the Balearic Islands.

OBJECTIVES: Newborn screening strategies for cystic fibrosis (CF) are run worldwide, and aim at the early detection of the disorder to significantly improve the quality of life. Elevated levels of immunoreactive trypsinogen (IRT) represent a high likelihood for the screened child to be affected with CF. However, the specificity of IRT is low. The objective of this study was to assess the screening program in the Balearic Islands during the past 14 years. DESIGN & METHODS: We evaluated all results of the screening program after 14 years, by considering all changes in the protocol and assessing the number of positive samples, the mutations detected, the number of sweat tests performed, the incidence of CF and the presence of false-negative cases. RESULTS: Despite a great variability among the different Balearic Islands, the global incidence of CF was 1:6059 for the 14 years assessed. The incidence in the smaller islands is about 5 times higher than in Majorca (1:2376 versus 1:10,613). After different changes in the protocol, an IRT cut-off value of 60 ng/mL was established. The two most common mutations are ΔF508 and G542X, in accordance with other geographical regions. CONCLUSIONS: The changes in the protocol helped reduce the number of sweat tests performed without any increase in the false-negative rate.

Clonal dissemination, emergence of mutator lineages and antibiotic resistance evolution in Pseudomonas aeruginosa cystic fibrosis chronic lung infection.

Chronic respiratory infection by Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF). We investigated the interplay between three key microbiological aspects of these infections: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of antibiotic resistance. For this purpose, 10 sequential isolates, covering up to an 8-year period, from each of 10 CF patients were studied. As anticipated, resistance significantly accumulated overtime, and occurred more frequently among mutator variants detected in 6 of the patients. Nevertheless, highest resistance was documented for the nonmutator CF epidemic strain LES-1 (ST-146) detected for the first time in Spain. A correlation between resistance profiles and resistance mechanisms evaluated [efflux pump (mexB, mexD, mexF, and mexY) and ampC overexpression and OprD production] was not always obvious and hypersusceptibility to certain antibiotics (such as aztreonam or meropenem) was frequently observed. The analysis of whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) revealed that a single genotype (clone FQSE-A) produced persistent infections in 4 of the patients. Multilocus Sequence typing (MLST) identified clone FQSE-A as the CF epidemic clone ST-274, but striking discrepancies between PFGE and MLST profiles were evidenced. While PFGE macrorestriction patterns remained stable, a new sequence type (ST-1089) was detected in two of the patients, differing from ST-274 by only two point mutations in two of the genes, each leading to a nonpreviously described allele. Moreover, detailed genetic analyses revealed that the new ST-1089 is a mutS deficient mutator lineage that evolved from the epidemic strain ST-274, acquired specific resistance mechanisms, and underwent further interpatient spread. Thus, presented results provide the first evidence of interpatient dissemination of mutator lineages and denote their potential for unexpected short-term sequence type evolution, illustrating the complexity of P. aeruginosa population biology in CF.

Exhaled Nitric Oxide in Acute Phase of Bronchiolitis and Its Relation with Episodes of Subsequent Wheezing in Children of Preschool Age.

BACKGROUND: Fractional exhaled nitric oxide (FENO) levels are increased in children with asthma and in infants with recurrent wheezing, but the role of FENO in the acute phase of bronchiolitis is still not defined. OBJECTIVE: The aim of this study is to evaluate FENO values in the acute phase of bronchiolitis, compare them with healthy infants, and relate those values with the appearance of other wheezing episodes. METHODS: FENO values were determined in infants between 2 months and 2 years affected with RVS bronchiolitis by offline method. The FENO values collected in the acute phase were related with the respiratory clinical symptoms presented in the 2 years following the episode. RESULTS: A total of 30 patients were recruited: 15 in the bronchiolitis group and 15 in the control group. The average of the FENO values in the acute phase was 18.74 ppb (range 2-88) in the bronchiolitis group, and 8.75 ppb (range 2-24) in the control group. However, these results showed no significant statistical differences (p=0.176). Nevertheless, we found a positive correlation between the FENO values and the clinical score (Downes) of the bronchiolitis episode (p=0.023). In infants that presented other wheezing episodes in the 2 years after, the average of FENO in the acute phase of the first episode was 23.1 ppb (average of 10.25 ppb) versus 8.4 ppb (average 5.4 ppb) in the group of patients with no other episodes. The comparison of averages has no statistical significance. CONCLUSION: We found no differences in FENO between infants with bronchiolitis and healthy ones. The FENO values in the acute phase seems to be related to the severity of the disease but do not predict the appearance of wheezing episodes in the following 2 years.

Risk of varicella complications in children treated with inhaled steroids.

We have studied the complications of varicella in children treated with inhaled steroids. From 1995 to 2005, 3/213 (1.4%) children hospitalized on the island of Mallorca, Spain, for varicella had received inhaled steroids. The rate of hospitalization for varicella complications in children receiving inhaled steroids was 17/100,000 children taking inhaled steroids, a rate similar to the rest of the children's population (18.2/100,000). Inhaled steroids did not increase the risk of varicella complications (relative risk 0.94; 95% confidence interval: 0.3-2.89).

[Clinical and epidemiological characteristics of respiratory infections caused by human metapneumovirus in pediatric patients]. / Características clínicas y epidemiológicas de las infecciones respiratorias causadas por el metapneumovirus humano en pacientes pediátricos.

INTRODUCTION: Human Metapneumovirus (hMPV) was first identified in 2001 in respiratory samples from children and adults with acute respiratory tract infection. The aim of this prospective study was to determine the clinical and epidemiological characteristics of pediatric patients with an acute respiratory tract infection and exclusive isolation of hMPV in respiratory samples (December 2005-January 2007). MATERIAL AND METHODS: All respiratory tract samples were submitted to rapid antigen detection against respiratory syncytial virus (RSV) and influenza A and B viruses. To isolate respiratory viruses, samples were inoculated in various cell lines using the shell vial culture assay. To isolate hMPV, the LLC-MK2 cell line was used. Only antigen-negative samples were studied for the presence of hMPV. RESULTS: Over the study period, 32 hMPV were isolated from different patients, accounting for 1.7% of all samples studied for this virus (only RSV-negative samples, 1,791) and 1.5% of all samples studied. Peak incidence was found between December and March of the two years studied. Of the 32 patients in whom hMPV was detected, 17 (53.2%) were female and 15 (46.8%) male. Mean age was 12.5 months (range, 1 month-4 years). The most frequent clinical symptoms were fever (90.6%), cough (87.5%), rhinorrhea and bronchiolitis (46.8%). Three patients (9.4%) were hospitalized. CONCLUSIONS: Respiratory infection caused by hMPV is considered an emergent disease in pediatric patients. The clinical manifestations are very similar to those of RSV infection; hence, only virological study can establish the definite etiological diagnosis.

Comparison of different cell lines and incubation times in the isolation by the shell vial culture of human metapneumovirus from pediatric respiratory samples.

We report a prospective study concerning the efficacy of LLC-MK2 (continuous monkey kidney cell), Hep-2, MDCK (Madin-Darby Canine Kidney), Vero and MRC-5 cell lines, by shell vial assay, and incubation time in the isolation of hMPV from pediatric respiratory samples. The overall sensitivity of the cell lines studied were: 100% for the LLC-MK2, 68.7% for the Hep-2, 28.1% for the Vero, 3.1% for the MDCK and 0% for the MRC-5. Only one strain (3.1%) showed growth in the four cell lines studied and 10 (31.2%) strains only grew in the LLC-MK2 cell line. The analysis of incubation times showed that only 14 strains (43.7%) were able to grow after 3 days of incubation, while all strains (100%) showed growth after 5 days. The use of shell vials with commercial LLC-MK2 cells could be a method for isolating hMPV from respiratory samples in the pediatric population.

Gammadelta T lymphocytes from cystic fibrosis patients and healthy donors are high TNF-alpha and IFN-gamma-producers in response to Pseudomonas aeruginosa.

BACKGROUND: Gammadelta T cells have an important immunoregulatory and effector function through cytokine release. They are involved in the responses to Gram-negative bacterium and in protection of lung epithelium integrity. On the other hand, they have been implicated in airway inflammation. METHODS: The aim of the present work was to study intracytoplasmic IL-2, IL-4, IFN-gamma and TNF-alpha production by gammadelta and alphabeta T lymphocytes from cystic fibrosis patients and healthy donors in response to Pseudomonas aeruginosa (PA). Flow cytometric detection was performed after peripheral blood mononuclear cells (PBMC) culture with a cytosolic extract from PA and restimulation with phorbol ester plus ionomycine. Proliferative responses, activation markers and receptor usage of gammadelta T cells were also evaluated. RESULTS: The highest production of cytokine was of TNF-alpha and IFN-gamma, gammadelta being better producers than alphabeta. No differences were found between patients and controls. The Vgamma9delta2 subset of gammadelta T cells was preferentially expanded. CD25 and CD45RO expression by the alphabeta T subset and PBMC proliferative response to PA were defective in cystic fibrosis lymphocytes. CONCLUSION: Our results support the hypothesis that gammadelta T lymphocytes play an important role in the immune response to PA and in the chronic inflammatory lung reaction in cystic fibrosis patients. They do not confirm the involvement of a supressed Th1 cytokine response in the pathogenesis of this disease.