Julian Legg

Frequency of detection of picornaviruses and seven other respiratory pathogens in infants.

Background: Previous studies in which molecular-based techniques have been used to identify the causative pathogens of respiratory tract infection have investigated hospitalized children only. We report a prospective study designed to determine the frequency and clinical presentation of community-acquired respiratory illness in infancy associated with 8 common respiratory pathogens. Methods: Eighty-eight infants were monitored through their first winter. With each respiratory illness, infants were examined, and a nasal lavage specimen was collected. Individual reverse transcription-polymerase chain reactions were performed to detect infection with picornaviruses (rhinoviruses and enteroviruses), coronaviruses (serotypes OC43 and 229E), adenoviruses, parainfluenza viruses 1–3, influenza viruses (types A and B), respiratory syncytial virus (RSV), Chlamydia pneumoniae and Mycoplasma pneumoniae. Results: Picornaviruses were the most frequently detected pathogen identified in 46% (56 of 123) of episodes, followed by RSV (27%), parainfluenza viruses (13%) and coronaviruses (9%). Dual pathogen infections were identified in 20% of episodes, predominantly caused by picornaviruses together with either RSV or parainfluenza viruses. RSV infection was significantly associated with a diagnosis of bronchiolitis. No other associations were found between pathogen and clinical diagnosis. Dual infection did not predispose infants to a more severe clinical course. Conclusions: Picornaviruses are the predominant cause of community-acquired respiratory tract infection in the first year of life. Large prospective community-based studies will be needed to fully evaluate the contribution of picornaviruses, both in isolation and in combination with other respiratory pathogens, to the various clinical syndromes of respiratory infection observed during infancy.

Impact of antibiotic treatment for pulmonary exacerbations on bacterial diversity in cystic fibrosis

BACKGROUND A diverse array of bacterial species is present in the CF airways, in addition to those recognised as clinically important. Here, we investigated the relative impact of antibiotics, used predominantly to target Pseudomonas aeruginosa during acute exacerbations, on other non-pseudomonal species. METHODS The relative abundance of viable P. aeruginosa and non-pseudomonal species was determined in sputa from 12 adult CF subjects 21, 14, and 7 days prior to antibiotics, day 3 of treatment, the final day of treatment, and 10-14 days afterwards, by T-RFLP profiling. RESULTS Overall, relative P. aeruginosa abundance increased during antibiotic therapy compared to other bacterial species; mean abundance pre-antibiotic 51.0±36.0% increasing to 71.3±30.4% during antibiotic (ANOVA: F(1,54)=5.16; P<0.027). Further, the number of non-pseudomonal species detected fell; pre-antibiotic 6.0±3.3 decreasing to 3.7±3.3 during treatment (ANOVA: F(1,66)=5.11; P<0.027). CONCLUSIONS Antibiotic treatment directed at P. aeruginosa has an additional significant impact on non-pseudomonal, co-colonising species.

Exhaled nitric oxide monitoring does not reduce exacerbation frequency or inhaled corticosteroid dose in paediatric asthma: a randomised controlled trial

Inhaled corticosteroid therapy (ICS) for asthma is currently modified according to symptoms and lung function. Fractional exhaled nitric oxide (FENO) has been demonstrated to be a non‐invasive marker of eosinophilic inflammation. Studies of FENO‐driven asthma management show variable success.

Molecular Microbiological Characterization of Preterm Neonates at Risk of Bronchopulmonary Dysplasia

The role of infection in bronchopulmonary dysplasia (BPD) is unknown. We present an observational study of 55 premature infants born weighing less than 1.3 kg within two level III neonatal intensive care units. Endotracheal aspirates (ETA) and nasogastric aspirates (NGA) were studied with denaturing gradient gel electrophoresis (DGGE) profiling to elucidate the total bacterial community, and species-specific PCR was used to detect the presence of Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. DGGE identified bacterial species in 59% of NGA and ETA samples combined. A diverse range of species were identified including several implicated in preterm labor. Species-specific PCR identified M. hominis in 25% of NGA and 11% of ETA samples. Among the 48 infants surviving up to 36 wk-postconceptual age, ordinal logistic regression showed the odds ratio for BPD or death where Ureaplasma was present/absent as 4.80 (95% CI 1.15–20.13). After adjusting for number of days ventilated, this was reduced to 2.04 (0.41–10.25). These data demonstrate how the combined use of DGGE and species-specific PCR identifies a high exposure in utero and around the time of birth to bacteria that might be causally related to preterm delivery and subsequent lung injury.

Low-dose nitric oxide as targeted anti-biofilm adjunctive therapy to treat chronic Pseudomonas aeruginosa infection in cystic fibrosis

Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases.

Update on the causes, investigation and management of empyema in childhood

Despite being recognised for over two millennia and having an increasing incidence in many countries, the management of childhood empyema remains controversial. This review examines the recent literature on its causes and investigation then focuses on its treatment. Following a trial of appropriate intravenous antibiotics, the evidence would currently support the use of chest drain insertion with urokinase instillation as first line treatment with video-assisted thoracoscopic surgery to be used for failure of medical management. A treatment algorithm is proposed to guide clinical practice.

Nitric oxide-mediated dispersal and enhanced antibiotic sensitivity in pseudomonas aeruginosa biofilms from the cystic fibrosis lung

Background and aims Bacterial biofilms present a major challenge in medicine due to their recalcitrance towards antimicrobials, relative to the same bacteria in a planktonic (free living) state. In cystic fibrosis (CF), bacteria colonise the lungs, establishing chronic and persistent infections which can not be eradicated with conventional antibiotic treatments. The effective treatment of CF patients colonised with the opportunistic pathogen Pseudomonas aeruginosa (PA) present a major therapeutic challenge not adequately addressed using current conventional antibiotic regimes (eg, ceftazadime and tobramycin in combination). We have previously demonstrated that biofilm formation by PA is reversed in vitro by nanomolar, non-toxic concentrations of NO by reduction of biofilm antibiotic tolerance. The aim of this project is to investigate nitric oxide as adjunctive therapy to standard antibiotic regimes in CF. Methods Biofilms from clinical isolates of PA were cultured ex vivo from sputum obtained from 20 teenagers and young adults with CF undergoing infective exacerbation. Dispersal, using low dose concentrations of the nitric oxide donor sodium nitroprusside (SNP), was calculated using optical density measurements. The effects of tobramycin and ceftazadime alone, and in combination with SNP, were visualised by Live/Dead fluorescent staining and confocal microscopy. Results The extent of biofilm dispersal was SNP-concentration dependent and could be significantly reduced by the addition of a nitric oxide scavenger. Moreover, biofilm dispersal was accompanied by increased susceptibility of PA to tobramycin and ceftazadime. Preliminary data also demonstrate dispersal and increased antibiotic susceptibility of multi-species biofilms cultured from CF sputum. Conclusions These data demonstrate that NO-mediated dispersal can augment the antibiotic sensitivity of PA biofilms from clinical isolates and have led to an ongoing NIHR Respiratory Biomedical Unit funded proof-of-principle clinical trial to assess the clinical role of nitric oxide as adjunctive therapy in CF teenagers and young adults colonised with PA (EudraCT 2010-023529-39).

A Quantitative Analysis of Ureaplasma urealyticum and Ureaplasma parvum Compared with Host Immune Response in Preterm Neonates at Risk of Developing Bronchopulmonary Dysplasia

ABSTRACT Multiplex, real-time PCR for the identification of Ureaplasma urealyticum and Ureaplasma parvum was performed on nucleic acids extracted from sequential endotracheal aspirates obtained from preterm neonates born at <29 weeks of gestation and ventilated for more than 48 h admitted to two level 3 neonatal intensive care units. Specimens were obtained shortly after birth and sequentially up until extubation. One hundred fifty-two specimens (93.8%) contained material suitable for analysis. Ureaplasma spp. were identified in 5 of 13 neonates studied. In most cases, the DNA load of the detected Ureaplasma species was low and decreased over time. In addition, changes in detectable Ureaplasma species DNA did not relate to changes in the inflammatory marker C-reactive protein (CRP) or respiratory status. All but two blood samples obtained at times of suspected sepsis were culture positive for other microorganisms; the species cultured were typically coagulase-negative staphylococci and were associated with increased levels of CRP (>10 mg/liter). This study was limited by the small number of patients examined and does not have the power to support or contradict the hypothesis that postnatal lung infection with Ureaplasma parvum is causally related to bronchopulmonary dysplasia (BPD) or adverse respiratory outcomes after preterm birth. However, in this study, increases in CRP levels were not associated with patients in whom Ureaplasma parvum was detected, in contrast to the detection of other bacterial species.

Asthma — The changing face of drug therapy

Until the recent introduction of long acting p2-agonists and the leukotriene antagonists, the drug treatment of asthma had remained largely unchanged for a quarter century. Recent studies have demonstrated the efficacy of the long acting pjagonists in the management of asthma in children and highlighted their value as an adjunct to inhaled corticosteroids. The leukotriene antagonists are an important new class of drug therapy which target a specific area of asthma pathogenesis. Whilst they have been shown to be effective for asthma, their exact role in the clinical situation remains to be established. Recent guidelines have emphasised the important role of inflammation in persistent asthma and recommended the early institution of anti-inflammatory treatment. Many patients remain uncontrolled despite high doses of anti-inflammatory agents including oral corticosteroids. Recent experience with other immunomodulatory agents such as cyclosporin, methotrexate and intravenous immunoglobulin has highlighed their potential as steroid sparing agents.With improved understanding of asthma pathogenesis the potential for specific targeted therapies has become evident. Monoclonal antibodies to IgE and certain cytokines are being investigated as possible treatments for asthma. Similarly, preliminary studies of selective phosphodiesterase inhibitors in asthmatic individuals have been encouraging. Other potential therapies include platelet-activating factor receptor antagonists, tryptase inhibitors and prostaglandin E analogs. The continued development of such targetea1 treatments should ensure a greater diversity of therapeutic options for the management of asthma in the new millennium.

Ciprofloxacin during upper respiratory tract infections to reduce Pseudomonas aeruginosa infection in paediatric cystic fibrosis: a pilot study

Objectives: Acute viral respiratory illnesses are associated with acquisition of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients. This study aimed to pilot a protocol for a randomized controlled trial to determine whether oral antipseudomonal antibiotics used at the onset of such episodes might delay onset of infection with this organism. Methods: A total of 41 children with CF aged 2–14 years, without chronic Pseudomonas infection, were randomized to receive ciprofloxacin (n = 28) or placebo (n = 13) at the onset of acute viral respiratory infections on an intention to treat basis, during a study period of up to 32 months. Results: There were no unexpected adverse events believed related to the use of the study medication. The rate of withdrawal from the study was low (approximately 7%) and did not differ between groups. Randomization was effective and acceptable to participants. Primary and secondary outcome measures all favoured active treatment, but there were no significant between group differences. The median rate of Pseudomonas isolates was 0/patient/year (interquartile range 0–0.38) in both the active and placebo groups. Kaplan–Meier survival curves showed no significant difference in time to first Pseudomonas isolate between groups. Conclusions: This study demonstrated the clinical feasibility of using oral ciprofloxacin in CF patients at times of viral infection. Within this sample size, no significant association was found between active treatment and decreased growth of Pseudomonas in follow-up microbiological samples. A definitive study would require at least 320 children to demonstrate significant differences in the rate of pseudomonal isolates.