Cystic Fibrosis Respiratory Specimens Research Articles

Clinically undetected polyclonal heteroresistance among Pseudomonas aeruginosa isolated from cystic fibrosis respiratory specimens.

Pseudomonas aeruginosa infection is the leading cause of death among patients with cystic fibrosis (CF) and a common cause of difficult-to-treat hospital-acquired infections. P. aeruginosa uses several mechanisms to resist different antibiotic classes and an individual CF patient can harbour multiple resistance phenotypes. To determine the rates and distribution of polyclonal heteroresistance (PHR) in P. aeruginosa by random, prospective evaluation of respiratory cultures from CF patients at a large referral centre over a 1 year period. We obtained 28 unique sputum samples from 19 CF patients and took multiple isolates from each, even when morphologically similar, yielding 280 unique isolates. We performed antimicrobial susceptibility testing (AST) on all isolates and calculated PHR on the basis of variability in AST in a given sample. We then performed whole-genome sequencing on 134 isolates and used a machine-learning association model to interrogate phenotypic PHR from genomic data. PHR was identified in most sampled patients (n = 15/19; 79%). Importantly, resistant phenotypes were not detected by routine AST in 26% of patients (n = 5/19). The machine-learning model, using the extended sampling, identified at least one genetic variant associated with phenotypic resistance in 94.3% of isolates (n = 1392/1476). PHR is common among P. aeruginosa in the CF lung. While traditional microbiological methods often fail to detect resistant subpopulations, extended sampling of isolates and conventional AST identified PHR in most patients. A machine-learning tool successfully identified at least one resistance variant in almost all resistant isolates by leveraging this extended sampling and conventional AST.

Is prolonged incubation required for optimal recovery of Burkholderia cepacia complex in sputum from cystic fibrosis patients? Data versus dogma

Cystic fibrosis (CF) expert groups globally recommend using selective medium for isolation of Burkholderia cepacia complex (BCC) from respiratory specimens of CF patients. However, there is no consensus available for optimal duration of incubation and recommendations are variable. The purpose of our study was to compare the difference in recovery of BCC in CF samples at 48 hours versus 7 days when inoculated on Burkholderia cepacia selective agar. A total of 307 consecutive clinical respiratory specimens from our local CF unit were studied prospectively (August 2017 to December 2017). All specimens were inoculated on Burkholderia cepacia medium, containing polymyxin B, gentamicin and ticarcillin. In our laboratory, these plates are routinely incubated for 48 hours as per the manufacturer's recommendation. However, for this study all plates with no growth at 48 hours were further incubated for total of 7 days at 35°C in O2. Plates were read daily to look for any growth. Microbial identification was performed using MALDI-TOF Vitek MS (database V3.0). Of the 307 CF respiratory specimens cultured, 177 (58%) were from paediatric and 130 (42%) were from adult patients; 155 (50%) specimens were sputum, 148 (48%) were cough swabs and four (1%) were bronchoalveolar lavage (BAL). All specimens from adults were sputum except one BAL. Thirteen (4%) cultures from eight adult and five paediatric specimens grew BCC. The majority (294, 96%) of specimens had no growth when incubated for 7 days. All 13 positive isolates recovered within 48 hours and there were no additional positive isolates found beyond 48 hours of incubation. We conclude from our analysis that prolonged incubation is not warranted for recovery of BCC in CF specimens if selective medium containing gentamicin and polymyxin is used. By adopting this approach of non-extended incubation, the burden of work on laboratory personnel can be significantly reduced and much faster turnaround time for CF cultures achieved. Our study confirms the results of recently published data on this point and challenges the prevailing dogma of utility of extended incubation for BCC isolation. For devising consensus statements for microbiology laboratories on this issue, CF societies and expert groups should consider reviewing data from the recent studies.

Toward the Standardization of Mycological Examination of Sputum Samples in Cystic Fibrosis: Results from a French Multicenter Prospective Study.

Fungal respiratory colonization of cystic fibrosis (CF) patients emerges as a new concern; however, the heterogeneity of mycological protocols limits investigations. We first aimed at setting up an efficient standardized protocol for mycological analysis of CF sputa that was assessed during a prospective, multicenter study: "MucoFong" program (PHRC-06/1902). Sputa from 243 CF patients from seven centers in France were collected over a 15-month period and submitted to a standardized protocol based on 6 semi-selective media. After mucolytic pretreatment, sputa were plated in parallel on cycloheximide-enriched (ACT37), erythritol-enriched (ERY37), benomyl dichloran-rose bengal (BENO37) and chromogenic (CAN37) media incubated at 37°C and on Sabouraud-chloramphenicol (SAB27) and erythritol-enriched (ERY27) media incubated at 20-27°C. Each plate was checked twice a week during 3weeks. Fungi were conventionally identified; time for detection of fungal growth was noted for each species. Fungal prevalences and media performances were assessed; an optimal combination of media was determined using the Chi-squared automatic interaction detector method. At least one fungal species was isolated from 81% of sputa. Candida albicans was the most prevalent species (58.8%), followed by Aspergillus fumigatus (35.4%). Cultivation on CAN37, SAB27, ACT37 and ERY27 during 16days provided an optimal combination, detecting C. albicans, A. fumigatus, Scedosporium apiospermum complex and Exophiala spp. with sensitivities of 96.5, 98.8, 100 and 100%. Combination of these four culture media is recommended to ensure the growth of key fungal pathogens in CF respiratory specimens. The use of such consensual protocol is of major interest for merging results from future epidemiological studies.

Assessing Airway Microbiota in Cystic Fibrosis: What More Should Be Done?

The use of culture-independent methods has deepened our appreciation of the complexity of the bacterial communities that typically reside in the airways of persons with cystic fibrosis (CF). New insights into how changes in the structure of these communities relate to lung disease progression will likely raise expectations for more in-depth microbiologic analysis of CF respiratory specimens. An article in this issue of the Journal of Clinical Microbiology (W. G. Flight, A. Smith, C. Paisey, J. R. Marchesi, M. J. Bull, P. J. Norville, K. J. Mutton, A. K. Webb, R. J. Bright-Thomas, A. M. Jones, and E. Mahenthiralingam, J Clin Microbiol 53:2022-2029, 2015, http://dx.doi.org/10.1128/JCM.00432-15) describes the application of a culture-independent approach to the assessment of CF airway microbiota.

Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients

Filamentous fungi cultured from respiratory tract specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were identified by morphology and by internal transcribed spacer (ITS) sequencing. Of 343 fungal isolates, discrepancies between identification methods were observed for four isolates (1.2%), while identification to species was achieved only with ITS sequencing for 16 isolates (4.7%). Filamentous fungi were isolated from 15% of cystic fibrosis (CF) respiratory samples in contrast to 2% of non-CF samples. From CF patients, a total of nine different species were found in 188 samples from 48 patients, whereas from non-CF patients, 24 different species were found in 155 samples from 111 patients. CF was associated with a significant overrepresentation of Aspergillus fumigatus and Scedosporium species; in contrast, the frequency of Penicillium spp. and other putative contaminants were significantly increased in non-CF patients. The altered species variation of filamentous fungi in CF respiratory specimens is contradictory to a scenario of incidentally inhaled spores, trapped in the viscous airway mucus of these patients and subsequently expectorated; rather, our data most likely reflect both an increased prevalence and an increased proportion of truly colonizing fungi in this patient group.

Low occurrence of ‘non-haemolytic Haemophilus haemolyticus’ misidentified as Haemophilus influenzae in cystic fibrosis respiratory specimens, and frequent recurrence of persistent H. influenzae clones despite antimicrobial treatment

Non-influenzae commensal Haemophilus species of low pathogenicity may be difficult to discriminate from Haemophilus influenzae. We investigated the level of misidentifications in respiratory specimens from cystic fibrosis patients and evaluated the colonisation dynamics of genuine H. influenzae isolates. One hundred and ninety-two presumptive H. influenzae isolates were re-examined by assessment of marker genes sodC and fucK, and isolates with aberrant genotypes were subjected to multilocus sequence typing. Misidentifications (3%) were mainly caused by failure to identify porphyrin-synthesising strains, and only a single strain (0.5%) could be classified as ‘non-haemolytic Haemophilus haemolyticus’. Sequential isolates of confirmed H. influenzae isolates from individual patients were typed by pulsed-field gel electrophoresis. Despite the routine prescription of antimicrobial therapy, the majority of H. influenzae isolates were identical with at least one of the strains cultured from the two preceding positive samples from the same patient.

Compliance of Clinical Microbiology Laboratories in the United States with Current Recommendations for Processing Respiratory Tract Specimens from Patients with Cystic Fibrosis

Respiratory tract specimens from patients with cystic fibrosis (CF) require unique processing by clinical microbiology laboratories to ensure detection of all potential pathogens. The present study sought to determine the compliance of microbiology laboratories in the United States with recently published recommendations for CF respiratory specimens. Microbiology laboratory protocols from 150 of 190 (79%) CF care sites were reviewed. Most described the use of selective media for Burkholderia cepacia complex (99%), Staphylococcus aureus (82%), and Haemophilus influenzae (89%) and identified the species of all gram-negative bacilli (87%). Only 52% delineated the use of agar diffusion assays for susceptibility testing of Pseudomonas aeruginosa. Standardizing laboratory practices will improve treatment, infection control, and our understanding of the changing epidemiology of CF microbiology.