Discrepancy in MALDI-TOF MS identification of uncommon Gram-negative bacteria from lower respiratory secretions in patients with cystic fibrosis (original) (raw)
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MALDI-TOF MS identification of bacteria from patients with CF
2015
Introduction: Early identification of microbial organisms from respiratory secretions of patients with cystic fibrosis (CF) is important to guide therapeutic decisions. Read this original research and sign up to receive Infection and Drug Resistance journal here: http://www.dovepress.com/articles.php?article\_id=21554
Molecular typing of the bacterial flora in sputum of cystic fibrosis patients
International Journal of Medical Microbiology, 2003
Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality. A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment. The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions. Sputa of 13 CF patients (7 males/6 females, age 19 ± 59 years) collected at the Stockholm CF centre were analysed. TTGE revealed the presence of complex bacterial profiles in all samples. The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA Pyrosequencing TM . DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora. The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports. However, the methodology seems too elaborate to be introduced into daily routine
Journal of Cystic Fibrosis, 2012
Identification of non-fermenting Gram-negative bacteria (NFGNB) from cystic fibrosis (CF) patients is often limited. A collection of stored NFGNB isolates (n = 182) recovered from CF patients over a 15 year period was examined. The routinely reported identification during this period was compared with that obtained by MALDI-TOF MS. Isolates giving discrepant identification at the genus level were further analyzed by 16S rDNA sequencing. The MALDI-TOF MS system identified 94% of the isolates, including Burkholderia cepacia and Pandoraea spp. isolates, the latter previously misidentified as other NFGNB by conventional microbiological methods. Lack of identification by MALDI-TOF MS was associated with the absence of entries in the database.
FEMS Immunology and Medical Microbiology, 2000
The nature of the micro-flora present in sputa of six different cystic fibrosis (CF) patients was assessed using routine microbiological culture and molecular methods. Bacterial genes for the small subunit ribosomal RNA (ssu rDNA) were specifically amplified from DNA extracted from the sputum samples, cloned and characterised by hybridisation and DNA sequencing. A large number of clones from six sputa were screened. Initially, oligonucleotide hybridisation was performed with five probes, specific for Gram-positives and Gramnegatives in general and the main pathogens for the CF patient (Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae). For a single sputum sample, the results were fully congruent when culture and molecular methods were compared. In the other five sputa, discrepancies for S. aureus and/or H. influenzae were documented. Although S. aureus DNA and H. influenzae DNA was detected in three and four sputa, respectively, strains could not be cultured. Although the PCR approach is not capable of distinguishing viable from dead bacteria, all of the CF patients had a history of S. aureus infections, while one of the CF patients once had cultivable H. influenzae in the sputum as well. A number of clones for probe-unidentified Gram-negative or Gram-positive bacterial species were further analysed by sequencing and additional potential pathogens were identified. Although routine culture of sputum frequently points to mono-specific exacerbations, our molecular data indicate that the other CF-related pathogens appear to be persistently present as well. We conclude that routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in a viable status in the lungs of these patients.
Changing Epidemiology of the Respiratory Bacteriology of Patients With Cystic Fibrosis
Chest, 2016
BACKGROUND: Monitoring potential changes in the epidemiology of cystic fibrosis (CF) pathogens furthers our understanding of the potential impact of interventions. METHODS: We performed a retrospective analysis using data reported to the Cystic Fibrosis Foundation Patient Registry (CFFPR) from 2006 to 2012 to determine the annual percent changes in the prevalence and incidence of selected CF pathogens. Pathogens included Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus (MSSA), methicillinresistant S aureus (MRSA), Haemophilus influenzae, Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans. Changes in nontuberculous mycobacteria (NTM) prevalence were assessed from 2010 to 2012, when the CFFPR collected NTM species. RESULTS: In 2012, the pathogens of highest prevalence and incidence were MSSA and P aeruginosa, followed by MRSA. The prevalence of A xylosoxidans and B cepacia complex were relatively low. From 2006 to 2012, the annual percent change in overall (as well as in most age strata) prevalence and incidence significantly decreased for P aeruginosa and B cepacia complex, but significantly increased for MRSA. From 2010 to 2012, the annual percent change in overall prevalence of NTM and Mycobaterium avium complex increased. CONCLUSIONS: The epidemiology of CF pathogens continues to change. The causes of these observations are most likely multifactorial and include improvements in clinical care and infection prevention and control. Data from this study will be useful to evaluate the impact of new therapies on CF microbiology.
Frontiers in Pediatrics, 2018
Purpose: Different respiratory sampling methods exist to identify lower airway pathogens in patients with cystic fibrosis (CF), of which bronchoalveolar lavage (BAL), and expectorated sputum are considered the "gold standard." Because BAL cannot be repeated limitless, the diagnosis of lower respiratory tract infections in non-expectorating patients is challenging. Other sampling techniques are nasal swab, cough swab, and induced sputum. The purpose of this study (NCT02363764) was to compare concordance between the microbiological yield of nasal swab, cough swab, and expectorated sputum in expectorating patients; nasal swab, cough swab, and induced sputum in non-expectorating patients; nasal swab, cough swab, induced sputum, and BAL in patients requiring bronchoscopy ("BAL-group"); and to determine the clinical value of cough swab in non-expectorating patients with CF. Methods: Microbiological yield detected by these different sampling techniques was compared between and within 105 expectorating patients, 30 non-expectorating patients and BAL-group (n = 39) in a single CF clinic. Specificity, sensitivity, positive (PPV), and negative (NPV) predictive values were calculated. Results: Overall low sensitivity (6.3-58.0%) and wide-ranging predictive values (0.0-100.0%) indicated that nasal swab was not appropriate to detect lower airway pathogens [Pseudomonas aeruginosa (Pa), Staphylococcus aureus (Sa), and Haemophilus influenzae (Hi)] in all three patient groups. Microbiological yield, specificity, sensitivity, PPV, and NPV of cough swab and induced sputum were largely similar in non-expectorating patients and in BAL-group (except sensitivity (0.0%) of induced sputum for Hi in BAL-group). Calculations for Pa and Hi could not be performed for non-expectorating patients because of low prevalence (n = 2 and n = 3, respectively). In expectorating patients, concordance was found between cough swab and expectorated sputum, except for Hi (sensitivity of 40.0%). Eyns et al. Respiratory Sampling in CF Conclusion: Our findings suggest that cough swab might be helpful in detecting the presence of some typical CF pathogens in the lower airways of clinically stable patients with CF. However, in symptomatic patients, who are unable to expectorate and who have a negative cough swab and induced sample, BAL should be performed as it currently remains the "gold standard."
A practical molecular identification of nonfermenting Gram-negative bacteria from cystic fibrosis
Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology], 2017
Identification of nonfermenting Gram-negative bacteria (NFGNB) of cystic fibrosis patients is hard and misidentification could affect clinical outcome. This study aimed to propose a scheme using polymerase chain reaction to identify NFGNB. This scheme leads to reliable identification within 3 days in an economically viable manner when compared to other methods.
Journal of Medical Microbiology, 2014
The predisposition of patients with cystic fibrosis (CF) for recurrent pulmonary infections can result in poor prognosis of the disease. Although the clinical significance in CF of microorganisms, such as Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa, is well established, the implication of uncommon glucose non-fermenting Gramnegative bacilli (UGNF-GNB) in respiratory samples from CF patients is still unclear. Because of limitations of traditional methods used in most clinical laboratories, the accurate identification of these microbes is a challenge. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) is an alternative tool for efficient identification of bacteria. This was a retrospective study to evaluate different identification methods in a collection of UGNF-GNB isolated from children with CF during a period of three years. The performance of MALDI-TOF was compared to that of 16S rDNA gene sequencing and to a conventional and automated phenotypic identification. The discriminatory power of MALDI-TOF (75.0 % agreement) was superior to automated techniques (67.1 % agreement) and to conventional phenotypical identification (50.0 % agreement). MALDI-TOF also demonstrated high accuracy in identifying Stenotrophomonas maltophilia, Achromobacter xylosoxidans and Chryseobacterium indologenes, but had limited utility in identifying Pandoraea spp. and some species of Acinetobacter and Chryseobacterium (other than C. indologenes). Although MALDI-TOF identified only 75 % of the isolates in comparison with 16S rDNA gene sequencing, the prompt identification and high discriminatory power exhibited by MALDI-TOF make it a useful tool for the characterization of micro-organisms that are difficult to identify using routine methods.