Biofilm on Toothbrushes of Children with Cystic Fibrosis: A Potential Source of Lung Re-Infection after Antibiotic Treatment?

Frequent recurrent lung infections result in irreversible lung damage in children with cystic fibrosis (CF). This study aimed to determine if toothbrushes contain biofilms of pathogens, and act as potential reservoirs for lung re-infection following antibiotic treatment of acute exacerbations. Toothbrushes were collected from children with CF of lung infection before, during and after antibiotic treatment. Toothbrushes were rinsed with sterile saline and cultured. Bacterial isolates from toothbrushes were identified by 16s rRNA gene sequencing and compared with isolates from a sputum sample of the same patient. Scanning electron microscopy (SEM) was used to visually confirm the presence of bacterial biofilms and confocal laser scanning microscopy (CLSM) combined with Live/Dead stain to confirm bacterial viability. Large numbers of bacteria and biofilms were present on all toothbrushes. SEM confirmed the presence of biofilms and CLSM confirmed bacterial viability on all toothbrushes. Pathogens identified on toothbrushes from children before and during antibiotics treatment were in concordance with the species found in sputum samples. Pseudomonas aeruginosa and Staphylococcus aureus was able to be cultured from children's toothbrushes despite antibiotic treatment. Toothbrushes were shown to be contaminated with viable pathogens and biofilms before and during antibiotic treatment and could be a potential source of lung re-infections.

Airway smooth muscle proliferation and inflammation in asthma.

In asthma, it is unclear if the airway smooth muscle cells proliferate more or are increased at the onset of asthma and remain stable. This study aimed to compare smooth muscle cell proliferation in individuals with and without asthma and correlate proliferation rates with cell size and number and with granulocytic airway inflammation. Postmortem airway sections were labeled with proliferating cell nuclear antigen (PCNA) and percent positive muscle cells calculated. On the same sections, smooth muscle cell size and number and the number of eosinophils and neutrophils were estimated and compared in cases of nonfatal ( n = 15) and fatal ( n = 15) asthma and control subjects ( n = 15). The %PCNA+ muscle cells was not significantly different in fatal (29.4 ± 7.7%, mean ± SD), nonfatal asthma (28.6 ± 8.3%), or control subjects (24.6 ± 6.7%) and not related to mean muscle cell size ( r = 0.09), number ( r = 0.36), thickness of the muscle layer ( r = 0.05), or eosinophil numbers ( r = 0.04) in the asthma cases. These data support the hypothesis that in asthma the increased thickness of the smooth muscle layer may be present before or at the onset of asthma and independent of concurrent granulocytic inflammation or exacerbation. NEW & NOTEWORTHY There is debate regarding the origins of the increased airway smooth muscle in asthma. It may be independent of inflammation or arise as a proliferative response to inflammation. The present study found no increase in the proportion of proliferating smooth muscle cells in asthma and no relation of proliferation to numbers of airway smooth muscle cells or inflammation. These results support a stable increase in smooth muscle in asthma that is independent of airway inflammation.

Inflammation-dependent and independent airway remodelling in asthma.

BACKGROUND AND OBJECTIVE: The pathology of asthma is characterized by airway inflammation (granulocytic (GA) or paucigranulocytic (PGA)) and remodelling of airway structures. However, the relationship between inflammatory phenotypes and remodelling is unclear. We hypothesized that some features of airway remodelling are dependent on granulocytic airway inflammation while others are not. METHODS: Post-mortem airway sections from control subjects (n = 48) and cases of asthma with (n = 51) or without (n = 29) granulocytic inflammation in the inner airway wall were studied. The thickness of the airway smooth muscle (ASM) layer, basement membrane and inner and outer airway walls, the size and number of ASM cells, the volume fraction of extracellular matrix within the ASM layer, ASM shortening and luminal mucus were estimated. Airway dimensions were compared between the three subject groups. RESULTS: In cases of PGA, only the thickness of the ASM layer and basement membrane was increased compared with control subjects. In cases of GA, not only the ASM and basement membrane were increased in thickness, but there was also increased inner and outer airway wall thickness and increased narrowing of the airway lumen due to ASM shortening and mucus obstruction, compared with control subjects. Granulocytic inflammation was observed more often in cases of fatal asthma. CONCLUSION: These findings suggest that inner and outer wall thickening coexists with inflammation, whereas thickening of the ASM layer and basement membrane may be present even in the absence of inflammation. Remodelling of the ASM layer and basement membrane may therefore be less susceptible to anti-inflammatory therapy.

Reproduction of post-weaning multi-systemic wasting syndrome in an animal disease model as a tool for vaccine testing under controlled conditions.

Snatch farrowed, colostrum deprived piglets were inoculated with different combinations of porcine circovirus 2, porcine parvovirus and Erysipelothrix rhusiopathiae candidate vaccines. 10 piglets were mock-vaccinated. Following virus challenge with a combined porcine circovirus 2/porcine parvovirus inoculum, all animals were monitored and samples taken for serology, immunohistochemistry and qPCR. At 24 dpc all non-vaccinated animals remaining were exhibiting signs of post-weaning multi-systemic wasting syndrome which was confirmed by laboratory analysis. Details of the study, analysis of samples and performance of the candidate vaccines are described.

Distribution of airway smooth muscle remodelling in asthma: relation to airway inflammation.

BACKGROUND AND OBJECTIVE: Pathological phenotypes of asthma have been based predominantly on inflammation, rather than airway wall remodelling. Differences in the distribution of airway smooth muscle (ASM) remodelling between large and small airways may affect clinical outcomes in asthma. The aim of this study was to examine the distribution of ASM remodelling and its relation to airway inflammation. METHODS: Post-mortem cases of asthma (n = 68) were categorized by the distribution of increased thickness of the ASM layer (relative to nonasthmatic controls, n = 37), into 'large only' (LO, n = 15), 'small only' (SO, n = 4) 'large/small' (LS, n = 24) or no increase (NI, n = 25). Subject characteristics, ASM and airway wall dimensions and inflammatory cell numbers were compared between groups. RESULTS: Apart from reduced clinical severity of asthma in NI cases (P = 0.002), subject characteristics did not distinguish asthma groups. Compared with control subjects, ASM cell number, reticular basement membrane thickness, airway wall thickness, percent muscle shortening and eosinophil number were increased (P < 0.05) in both large and small airways in LS cases and only the large airways in LO cases. Increased numbers of neutrophils were observed only in the small airways of LO cases. CONCLUSIONS: Distinct distributions of ASM remodelling are seen in asthma. Pathology limited to the small airways was uncommon. Increased thickness of the ASM layer was associated with airway remodelling and eosinophilia, but not neutrophilia. These data support the presence of distinct pathological phenotypes based on the site of increased ASM.

Safety of inhaled (Tobi®) and intravenous tobramycin in young children with cystic fibrosis.

BACKGROUND: Use of inhaled tobramycin therapy for treatment of Pseudomonas aeruginosa infections in young children with cystic fibrosis (CF) is increasing. Safety data for pre-school children are sparse. METHODS: The aim of this study was to assess the safety of tobramycin solution for inhalation (TOBI®-TSI) administered twice daily for 2 months/course concurrently to intravenous (IV) tobramycin during P. aeruginosa eradication therapy in children (0-5 years). Audiological assessment and estimation of glomerular filtration rate (GFR) was measured prior to any exposure and end of the study. RESULTS: Data were available from 142 patients who were either never exposed to aminoglycosides (n=39), exposed to IV aminoglycosides only (n=36) or exposed to IV+TSI (n=67). Median exposure to TSI was 113 days [59, 119]. Comparison of effects on audiometry results and GFR, showed no detectable difference between the groups. CONCLUSIONS: Use of TSI and IV tobramycin in pre-school children with CF was not associated with detectable renal toxicity or ototoxicity.

Detection of viral and bacterial respiratory pathogens in patients with cystic fibrosis.

The presence of viral respiratory infections is associated closely with exacerbations in patients with cystic fibrosis. Viral and bacterial multiplex PCRs were developed and applied to nasal swab samples from children with cystic fibrosis. This showed a large number of individuals with cystic fibrosis were infected with rhinoviruses, and more were infected with viral than bacterial pathogens. All individuals with parainfluenza 3 virus had clinical exacerbations of their cystic fibrosis, and although 3/4 of these children were co-infected with HRV. The findings do not suggest a significant association for any other virus or bacteria with exacerbation. There is clear evidence some viral infections are associated with cystic fibrosis that dual infection is more likely to produce symptoms, and mechanisms of viral-induced exacerbation should be elucidated.

Airway smooth muscle hypertrophy and hyperplasia in asthma.

RATIONALE: Increased thickness of the airway smooth muscle (ASM) layer in asthma may result from hyperplasia or hypertrophy of muscle cells or increased extracellular matrix (ECM). OBJECTIVES: To relate ASM hypertrophy, ASM hyperplasia, and deposition of ECM to the severity and duration of asthma. METHODS: Airways from control subjects (n = 51) and from cases of nonfatal (n = 49) and fatal (n = 55) asthma were examined postmortem. Mean ASM cell volume (V(C)), the number of ASM cells per length of airway (N(L)), and the volume fraction of extracellular matrix (f(ECM)) within the ASM layer were estimated. Comparisons between subject groups were made on the basis of general linear regression models. MEASUREMENTS AND MAIN RESULTS: Mean V(C) was increased in the large airways of cases of nonfatal asthma (P = 0.015) and fatal asthma (P < 0.001) compared with control subjects. N(L) was similar in nonfatal cases and control subjects but increased in large (P < 0.001), medium (P < 0.001), and small (P = 0.034) airways of cases of fatal asthma compared with control subjects and with nonfatal cases (large and medium airways, P ≤ 0.003). The f(ECM) was similar in cases of asthma and control subjects. Duration of asthma was associated with a small increase in N(L). CONCLUSIONS: Hypertrophy of ASM cells occurs in the large airways in both nonfatal and fatal cases of asthma, but hyperplasia of ASM cells is present in the large and small airways in fatal asthma cases only. Both are associated with an absolute increase in ECM. Duration of asthma has little or no effect on ASM hypertrophy or hyperplasia or f(ECM).

Improved survival in cystic fibrosis patients diagnosed by newborn screening compared to a historical cohort from the same centre.

BACKGROUND: Newborn screening (NBS) for cystic fibrosis (CF) is associated with improved early nutritional outcomes and improved spirometry in children. The aim of this study was to determine whether early diagnosis and treatment of CF with NBS in New South Wales in 1981 led to better clinical outcomes and survival into early adulthood. METHODS: Retrospective observational study comprising two original cohorts born in the 3 years before ('non-screened cohort', n=57) and after ('screened'; n=60) the introduction of NBS. Patient records were assessed at transfer from paediatric to adult care by age 19 years and survival was documented to age 25 years. RESULTS: Non-screened patients (n=38) when compared with screened patients (n=41) had a higher rate and lower age of Pseudomonas aeruginosa acquisition at age 18 years (p ≤ 0.01). Height, weight and body mass index (BMI) z scores (all p<0.01) and forced expiratory volume in 1 s (FEV(1))% were better in the screened group (n=41) (difference: 16.7 ± 6.4%; p=0.01) compared to non-screened (n=38) subjects on transfer to adult care. Each 1% increase in FEV(1)% was associated with a 3% (95% CI 1% to 5%; p=0.001) decrease in risk of death and each 1.0 kg/m(2) increase in BMI contributed to a 44% (95% CI 31% to 55%; p<0.001) decrease in risk of death. This accumulated in a significant survival difference at age 25 years (25 vs 13 deaths or lung transplants; p=0.01). CONCLUSION: NBS for CF leads to better lung function, nutritional status and improved survival in screened patients in early adulthood.

Cystic fibrosis in Australia, 2009: results from a data registry.

OBJECTIVES: To describe the demographics, clinical features and outcomes among people with cystic fibrosis (CF) in Australia and to estimate incidence of the disease. DESIGN AND SETTING: Cross-sectional analysis using data from the Australian Cystic Fibrosis Data Registry for 2009. MAIN OUTCOME MEASURES: Numbers of diagnoses, pulmonary and anthropometric measurements, microbiological culture results, rates of hospitalisation and transplantation, and numbers of medical complications and deaths. RESULTS: In 2009, data were submitted on 2986 people (48% female). Median age was 17.6 years and 49% of people were aged 18 years or over. Seventy-eight people were newly diagnosed. Fourteen people died and 14 people underwent lung transplantation in the year. Lung function and nutrition were relatively normal among children but deteriorated (more rapidly) among adolescents. With increasing age, progressive respiratory disease was apparent, and the frequency of CF-related complications and use of health care resources increased. In all age groups, there was a wide range in severity of lung disease and nutritional status. CONCLUSIONS: CF remains a progressive respiratory disease and is associated with multisystem complications. The acceleration in disease severity in adolescence and early adulthood suggests that better treatment at these stages is required to further improve survival.

Post-natal corticosteroids are associated with reduced expiratory flows in children born very preterm.

AIM: Infants born very prematurely often received corticosteroids to minimise the risk of developing bronchopulmonary dysplasia (BPD) but their long term impact on lung function at school age is unclear. METHODS: A cross-sectional study of 105 children [mean gestation of 27 weeks] was undertaken. Lung function assessments were conducted at a mean age of 10 years according to standard criteria. Corticosteroid dose was obtained from the medical record. RESULTS: Spirometry in the BPD group was not significantly different to the non-BPD group, mean per-cent predicted (95% confidence interval) forced expiratory volume in 1 s (FEV1) 83% (79, 87) versus 86% (83, 90), FEF25%-75% 67% (60, 73) versus 75% (69, 81). Antenatal steroid treatment alone did not adversely affect airflow FEV1, 88% (84.92) versus 90% (82.97), and forced expiratory flow (FEF)25%-75%, 75% (69.81) versus 87% (70.104). Children who received post-natal corticosteroids had significantly lower flows than those who did not (FEV1 82% (78.85) vs. 88% (85.92), P = 0.006; FEF25%-75% 65% (59.71) vs. 78% (72.84), P = 0.003). Regression analysis revealed days on oxygen and days ventilated were statistically significant but weak predictors of airflow at 10 years of age. CONCLUSIONS: A diagnosis of BPD did not predict reduced spirometry in middle childhood. Children who received post-natal corticosteroids as preterm infants had reduced expiratory flows compared with those who did not. While post-natal corticosteroids may be a marker of severity of lung disease, the potential of post-natal corticosteroids to influence lung development requires further investigation.

Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland.

We report the isolation in cell cultures of two novel bocavirus species in pigs from farms in Northern Ireland with clinical postweaning multisystemic wasting syndrome (PMWS). We have designated the isolates as porcine bocavirus-3 (PBoV3) and porcine bocavirus-4 (PBoV4). To date 5082 and 4125 bps of PBoV3 and PBoV4 have been sequenced, respectively. PBoV3 and PBoV4 show nucleotide homology to other known bocaviruses in swine and other organisms. Open reading frame (ORF) analysis has shown that these viruses have a third small ORF, equivalent to the NP1 ORF that distinguishes the bocaviruses from other parvoviruses. A panel of porcine field sera was screened by indirect immunofluorescence against both viruses. Of the 369 samples analysed, 32 (8.7%) and 35 (9.5%) sera were seropositive for PBoV3 and PBoV4 respectively, thus providing serological evidence of the exposure of swine in the field to bocavirus-like viruses. To date, the clinico-pathological significance of these novel swine bocaviruses, as primary pathogens or as immunosuppresive triggers for other infectious agents, is undetermined.

Feather bedding and childhood asthma associated with house dust mite sensitisation: a randomised controlled trial.

INTRODUCTION: Observational studies report inverse associations between the use of feather upper bedding (pillow and/or quilt) and asthma symptoms but there is no randomised controlled trial (RCT) evidence assessing the role of feather upper bedding as a secondary prevention measure. OBJECTIVE: To determine whether, among children not using feather upper bedding, a new feather pillow and feather quilt reduces asthma severity among house dust mite (HDM) sensitised children with asthma over a 1-year period compared with standard dust mite avoidance advice, and giving children a new mite-occlusive mattress cover. DESIGN: RCT. SETTING: The Calvary Hospital in the Australian Capital Territory and the Children's Hospital at Westmead, Sydney, New South Wales. PATIENTS: 197 children with HDM sensitisation and moderate to severe asthma. Intervention New upper bedding duck feather pillow and quilt and a mite-occlusive mattress cover (feather) versus standard care and a mite-occlusive mattress cover (standard). MAIN OUTCOME MEASURES: The proportion of children reporting four or more episodes of wheeze in the past year; an episode of speech-limiting wheeze; or one or more episodes of sleep disturbance caused by wheezing; and spirometry with challenge testing. Statistical analysis included multiple logistic and linear regression. RESULTS: No differences between groups were found for primary end points--frequent wheeze (OR 1.51, 95% CI 0.83 to 2.76, p=0.17), speech-limiting wheeze (OR 0.70, 95% CI 0.32 to 1.48, p=0.35), sleep disturbed because of wheezing (OR 1.17, 95% CI 0.64 to 2.13, p=0.61) or for any secondary end points. Secondary analyses indicated the intervention reduced the risk of sleep being disturbed because of wheezing and severe wheeze to a greater extent for children who slept supine. CONCLUSION: No differences in respiratory symptoms or lung function were observed 1 year after children with moderate-severe asthma and HDM sensitisation were given a mite-occlusive mattress cover and then received either feather upper bedding (pillow and quilt) or standard bedding care.

Home IV Antibiotic Therapy and Exercise Capacity in Children with CF: A Case Series.

PURPOSE: This case series describes the effect of home intravenous (IV) antibiotic therapy on spirometry and exercise capacity in a group of children with cystic fibrosis (CF). METHODS: Outcomes from 10 children with CF who were prescribed a 14-day course of home IV antibiotics for a respiratory exacerbation are reported. All children performed spirometry and a modified shuttle test (MST) before and after 14-days of home IV therapy. RESULTS: After 14 days, FEV(1) increased by mean (± SE) 12 ± 4 % (p < 0.05) but mean MST did not improve compared to baseline. All children improved or maintained spirometry values with treatment, however, only 5 improved MST distance. CONCLUSION: After 14 days of home IV antibiotic therapy, a significant improvement in spirometry, but not exercise capacity, was seen in this small series of children with CF. The lack of improvement in exercise capacity for all children following home IV antibiotic therapy suggests factors other than spirometry determine exercise capacity. Identifying and investigating the factors that influence exercise capacity during home IV antibiotic therapy requires further investigation.

Development of the bronchial epithelial reticular basement membrane: relationship to epithelial height and age.

BACKGROUND: The bronchial epithelium and underlying reticular basement membrane (RBM) have a close spatial and functional inter-relationship and are considered an epithelial-mesenchymal trophic unit (EMTU). An understanding of RBM development is critical to understanding the extent and time of appearance of its abnormal thickening that is characteristic of asthma. METHODS: RBM thickness and epithelial height were determined in histological sections of cartilaginous bronchi obtained postmortem from 47 preterm babies and infants (median age 40 weeks gestation (22 weeks gestation-8 months)), 40 children (2 years (1 month-17 years)) and 23 adults (44 (17-90) years) who had died from non-respiratory causes, and had no history of asthma. RESULTS: The RBM was visible by light microscopy at 30 weeks gestation. RBM thickness increased in successive age groups in childhood; in infants (r=0.63, p<0.001) and in children between 1 month and 17 years (r=0.82, p<0.001). After 18 years, RBM thickness decreased with increasing age (r=-0.42, p<0.05). Epithelial height showed a similar relationship with age, a positive relationship from preterm to 17 years (r=0.50, p<0.001) and a negative relationship in adulthood (r=-0.84, p<0.0001). There was a direct relationship between epithelial height and RBM thickness (r=0.6, p<0.001). CONCLUSIONS: The RBM in these subjects was microscopically identifiable by 30 weeks gestation. It thickened during childhood and adolescence. In adults, there was either no relationship with age, or a slow reduction in thickness in older age. Developmental changes of RBM thickness were accompanied by similar changes in epithelial height, supporting the close relationship between RBM and epithelium within the EMTU.

Normal development of the lung and premature birth.

The following review focuses on the normal development of the lung from conception to birth. The defined periods of lung development-Embryonic, Pseudoglandular, Canalicular, Saccular and Alveolar-will be explored in detail in relation to gestational age. Cellular differentiation, formation of the conducting airways and respiratory zone and development of the alveoli will be reviewed. Pulmonary vascular development will also be examined within these periods to relate the formation of the blood-air barrier to the lungs for their essential function of gas exchange after birth. The development of the surfactant and cortisol systems will also be discussed as these need to be mature before the lungs are able to take on their role of respiration following birth. It is clear that premature birth interrupts normal lung development so the effect of preterm birth on lung development will be examined and the respiratory consequences of very preterm birth will be briefly explored.

Segregation of children with CF diagnosed via newborn screening and acquisition of Pseudomonas aeruginosa.

BACKGROUND: Data from the Wisconsin newborn screening (NBS) study show that neonatally diagnosed infants are at risk of early Pseudomonas aeruginosa (PsA) acquisition. We have had NBS since 1981 and in 2003, introduced PsA-free 'segregation' from older patients for children < or =5. This study investigated the effect of simple 'segregation' on acquisition of respiratory pathogens. METHODS: Sputum culture results (n=2814) and details of antibiotic use before (1999-2002) and after (2004-2007) 'segregation' were collected. RESULTS: Each year each child provided an average of 4.6 samples for culture. There was a significant decrease (p< or =0.001 Chi(2)) in the acquisition of mucoid (from 5.9% of children to 1.0%) but not non-mucoid PsA (22.3% and 22.7%, respectively) after 'segregation'. There was no significant change in other respiratory pathogens. CONCLUSIONS: Young children with CF diagnosed via NBS can be protected from the acquisition of mucoid PsA by 'segregation' and the acquisition of non-mucoid PsA is likely to be from environmental sources outside the hospital.