Jackie Rendall

Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis

This study compares conventional and molecular techniques for the detection of fungi in 77 adult cystic fibrosis (CF) patients. Three different methods were investigated, i.e., (1) conventional microbiological culture (including yeasts and filamentous fungi), (2) mycological culture with CF-derived fungal specific culture media, and (3) Non-culture and direct DNA extraction from patient sputa. Fungi isolated from environmental air samples of the CF unit were compared to fungi in sputa from CF patients. Fungi (n = 107) were detected in 14/77(18%) of patients by method 1, in 60/77 (78%) of patients by method 2 and with method 3, in 77/77(100%) of the patients. The majority of yeasts isolated were Candida albicans and C. dubliniensis. Exophiala (Wangiella) dermatitidis, Scedosporium apiospermum, Penicillium spp., Aspergillus fumigatus, and Aspergillus versicolor were also identified by sequence analysis of the rDNA short internal transcribed spacer (ITS2) region. Conventional laboratory analysis failed to detect fungi in 63 patients mainly due to overgrowth by Gram-negative organisms. Mycological culture with antibiotics dramatically increased the number of fungi that could be detected. Molecular techniques detected fungi such as Saccharomyces cerevisiae, Malassezia spp., Fuscoporia ferrea, Fusarium culmorum, Acremonium strictum, Thanatephorus cucumeris and Cladosporium spp. which were not found with other methods. This study demonstrates that several potentially important fungi may not be detected if mycological culture methods alone are used. A polyphasic approach employing both enhanced mycological culture with molecular detection will help determine the presence of fungi in the sputa of patients with CF and their healthcare environment.

Fungal infections in patients with cystic fibrosis

Current US Cystic Fibrosis Foundation (CFF) Registry data indicates an approximate doubling of prevalence of fungi being detected in the sputum of patients with cystic fibrosis (CF), probably due to a better understanding of the disease and the way in which it is managed. The introduction of new antibiotics and the way in which they are used – in double or triple therapy and as well as in antibiotic cycling – has helped address the requirement to control chronic Gram-negative infections in CF. However, this has inadvertently created a niche for the colonization and proliferation of fungi in the CF lung. The ability to detect and isolate fungi in the sputum of CF patients is important due to the emerging significance of these organisms, particularly in relation to: (i) allergic bronchopulmonary aspergillosis (ABPA); (ii) post-transplant fungaemia; and (iii) chronic colonization/infection (bronchitis). Recent data has indicated that ABPA affects approximately 1–15% of CF patients and is associated with an accelerated decline in lung function. Most of the mycological aetiology of this condition is due to Aspergillus fumigatus and other Aspergillus spp, although other fungi have been reported. In addition, patients who have undergone lung transplantation are at increased risk of opportunistic fungal infection, such as invasive pulmonary aspergilloma and other invasive mycoses, due to the immunosuppression of the host. This review summarizes the important fungal pathogens in CF, namely Aspergillus spp., as well as the emerging fungi, including Scedosporium apiospermum and Exophiala dermatitidis and discusses the various conventional and molecular methods of detection and identification.

Molecular characterization of macrolide resistance determinants [erm(B) and mef(A)] in Streptococcus pneumoniae and viridans group streptococci (VGS) isolated from adult patients with cystic fibrosis (CF)

OBJECTIVES Although long-term use of azithromycin has shown a significant clinical improvement for patients with cystic fibrosis (CF), its long-term effect on the susceptibility of commensal flora within CF airways has not yet been examined. We therefore suggest that long-term use of azithromycin increases macrolide resistance in commensal streptococci. METHODS Erythromycin susceptibility in naturally colonizing viridans group streptococci (VGS) was characterized, as well as macrolide resistance gene determinants through sequence analysis, in pneumococci (n = 15) and VGS [n = 84; i.e. Streptococcus salivarius (n = 30), Streptococcus mitis (n = 17), Streptococcus sanguinis (n = 11), Streptococcus oralis (n = 10), Streptococcus parasanguinis (n = 6), Streptococcus gordonii (n = 3), Streptococcus infantis (n = 3), Streptococcus cristatus (n = 2), Streptococcus anginosus (n = 1) and Streptococcus australis (n = 1)] isolated from sputum from 24 adult CF patients, who were on oral azithromycin therapy for at least the previous 7 months. RESULTS Almost three-quarters of isolates (74; 74.7%) were resistant to erythromycin, whilst a further 15 (15.2%) had reduced susceptibility, leaving only 10 (10.1%) isolates susceptible to erythromycin. The majority (89.8%) were not susceptible to erythromycin, as demonstrated by possession of the erm(B) gene in 25/99 (25.3%), the mef(A) gene in 1/99 (1.0%), the mef(E) gene in 75/99 (75.8%) and both erm(B) and mef(E) genes simultaneously in 11/99 (11.1%). These results indicate that genotypic resistance for macrolides is common in VGS in adult CF patients, with efflux being over three times more frequent. CONCLUSIONS Long-term treatment with azithromycin in CF patients may reduce antibiotic susceptibility in commensal VGS, where these organisms may potentially act as a reservoir of macrolide resistance determinants for newly acquired and antibiotic-susceptible pathogens.

Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF)

Yeasts and filamentous fungi are beginning to emerge as significant microbial pathogens in patients with cystic fibrosis (CF), particularly in relation to allergic-type responses, as seen in patients with allergic bronchopulmonary aspergillosis (ABPA), Aspergillus bronchitis and in invasive fungal disease in lung transplant patients. Four fungal media were compared in this study, including Sabouraud Dextrose Agar (SDA) and Medium B, with and without the addition of selective antibiotics, where antibiotic-supplemented media were designated with (+). These media were compared for their ability to suppress contaminating, mainly Gram-ve pathogens, in CF sputa (Pseudomonas aeruginosa, Burkholderia cepacia complex [BCC] organisms) and to enhance the growth of fungi present in CF sputum. Medium B consisted of glucose (16.7 g/l), agar (20 g/l), yeast extract (30 g/l) and peptone (6.8 g/l) at pH 6.3 and both SDA(+) and Medium B(+) were supplemented with cotrimethoxazole, 128 mg/l; chloramphenicol, 50 mg/l; ceftazidime, 32 mg/l; colistin, 24 mg/l). Employment of SDA(+) or Medium B(+) allowed an increase in specificity in the detection of yeasts and moulds, by 42.8% and 39.3%, respectively, over SDA when used solely. SDA(+) had a greater ability than Medium B(+) to suppress bacterial growth from predominantly Gram-ve co-colonisers. This is a significant benefit when attempting to detect and isolate fungi from the sputum of CF patients, as it largely suppressed any bacterial growth, with the exception of the BCC organisms, thus allowing for an increased opportunity to detect target fungal organisms in sputum and represented a significant improvement over the commercial medium (SDA), which is currently used. Overall, both novel selective media were superior in their ability to suppress bacteria in comparison with the commercially available SDA medium, which is routinely employed in most clinical microbiology diagnostic laboratories presently. Alternatively, Medium B(+) had a great ability to grow fungi than SDA(+) and when employed together, the specificity of combined use was 82%, with a sensitivity for yeasts, filamentous fungi, and combined overall fungi of 96.0%, 92.3% and 96.0%, respectively. Overall, when employing one fungal selective medium for the routine detection of yeasts and filamentous fungi in the sputum of CF patients, we would recommend employment of Medium B(+). However, we would recommend the combined employment of SDA(+) and Medium B(+), in order to synergistically isolate and detect the greatest number of fungi present in CF sputa.

Temocillin in cystic fibrosis : a retrospective pilot study

BACKGROUND Temocillin is currently used in the treatment of acute pulmonary exacerbations caused by Burkholderia cepacia complex and multi-resistant Pseudomonas aeruginosa in cystic fibrosis (CF) patients despite little published clinical data. This study assessed if intravenous (IV) antibiotic therapy including temocillin was equivalent to standard combination therapy for an acute exacerbation. METHODS A retrospective, pilot cross-over study. Adult patients attending two CF centres between 1997 and 2006 who had received a course of IV antibiotics including temocillin (TIV) and a further IV course (within +/-1 year) which did not include temocillin (NTIV) were included. Outcome measures at the start and end of each IV course were recorded (FEV(1)%, FVC%). RESULTS Twenty six patients had received temocillin. Baseline values of FEV(1)% predicted were comparable for both groups (TIV: 37(18%), NTIV: 39(20%)). FEV(1)% increased by 7.12(11.67)% after TIV (p<0.01) and 6.65(7.62)% after NTIV (p<0.01). There was no significant difference between the IV courses in mean %change in lung function TIV versus NTIV (FEV(1) 0.46% [95%CI: -4.55 to 5.48%]). CONCLUSION These data suggest equivalence in the lung function outcome of IV antibiotic therapy includingtemocillin versus standard IV antibiotic therapy.

Multicentre trial of weekly risedronate on bone density in adults with cystic fibrosis

BACKGROUND The aim of this study was to assess the efficacy, tolerability and safety of risedronate in adults with CF. METHODS Patients with a lumbar spine (LS), total hip (TH) or femoral neck (FN) bone mineral density (BMD) Z-score of -1 or less were randomised to receive risedronate 35 mg weekly or placebo, and calcium (1g)+vitamin D(3) (800IU). RESULTS At baseline, BMD Z-scores in the risedronate (n=17) and placebo (n=19) groups were similar. By 24 months, 7/17 risedronate patients vs 0/19 placebo patients stopped the study medication due to bone pain. After 24 months treatment, the mean difference (95% CI) in change in LS, TH and FN BMD between the risedronate vs placebo groups was 4.3% (0.4, 8.2) p=0.03; 4.0% (-0.5, 8.6) p=0.08; and 2.4% (-3.5, 8.2) p=0.41. CONCLUSIONS After two years treatment there was a significant increase in LS BMD with weekly risedronate compared to placebo.

Isolation of Burkholderia cenocepacia and Burkholderia vietnamiensis from human sewage

Fresh human sewage was examined from a sewage treatment plant for the presence of members of the Burkholderia cepacia complex (BCC) of bacterial organisms and confirmed the presence of viable B. cenocepacia and B. vietnamiensis, by a combination of cultural, phenotypic and genotypic techniques. Both these organisms are important respiratory pathogens for patients with cystic fibrosis (CF). Presently, the survival dynamics of these organisms in sewage effluent and sludge is, as yet, unknown. Therefore, as this study represents the first report of these CF pathogens in sewage and until such survival data is available, careful risk assessment needs to be undertaken in relation to the end use application of potentially contaminated sewage and where such material comes into association with non-colonised patients with cystic fibrosis, so that any potential transmission of these pathogens from sewage to patient is assessed and minimised/eliminated.

Multicentre randomised double blind placebo controlled trial assessing the effect of weekly risedronate on bone mineral density in adults with cystic fibrosis

328* Multicentre randomised double blind placebo controlled trial assessing the effect of weekly risedronate on bone mineral density in adults with cystic fibrosis C.S. Haworth1, L. Sharples2, V. Hughes3, S.L. Elkin4, M.E. Hodson5, S.P. Conway6, C. Etherington6, J.S. Elborn7, J. Rendall7, E. Wheaton2, E. Kadri8, J. Elliott1, H.C. Barker1, J. Compston9. 1Papworth Hospital, Adult Cystic Fibrosis Centre, Cambridge, United Kingdom; 2Institute of Public Health, MRC Biostatistics Unit, Cambridge, United Kingdom; 3Papworth Hospital, Research and Development, Cambridge, United Kingdom; 4St Mary’s Hospital, Respiratory Medicine, London, United Kingdom; 5Royal Brompton Hospital, Department of Cystic Fibrosis, London, United Kingdom; 6St James’s University Hospital, Cystic Fibrosis Centre, Leeds, United Kingdom; 7Belfast City Hospital, Adult Cystic Fibrosis Centre, Belfast, United Kingdom; 8Papworth Hospital, Pharmacy, Cambridge, United Kingdom; 9University of Cambridge, Department of Medicine, Cambridge, United Kingdom

Emergence of Scedosporium apiospermum in patients with cystic fibrosis

The ubiquitous Scedosporium apiospermum is a saprophytic filamentous fungus that causes a wide range of human infections that affect virtually every organ in the body. S apiospermum has been described as one of the major fungal agents …