Altered Sputum Microstructure as a Marker of Airway Obstruction in Cystic Fibrosis Patients

NASA Astrophysics Data System (ADS)

Duncan, Gregg; Jung, James; West, Natalie; Boyle, Michael; Suk, Jung Soo; Hanes, Justin

In the lungs of cystic fibrosis (CF) patients, highly viscoelastic mucus remains stagnant in the lung leading to obstructed airways prone to recurrent infections. Bulk-fluid rheological measurement is primarily used to assess the pathological features of mucus. However, this approach is limited in detecting microscopic properties on the length scale of pathogens and immune cells. We have shown in prior work based on the transport of muco-inert nanoparticles (MIP) in CF sputum that patients can carry significantly different microstructural properties. In this study, we aimed to determine the factors leading to variations between patients in sputum microstructure and their clinical implications. The microrheological properties of CF sputum were measured using multi-particle tracking experiments of MIP. MIP were made by grafting polyethylene glycol onto the surface of polystyrene nanoparticles which prior work has shown prevents adhesion to CF sputum. Biochemical analyses show that sputum microstructure was significantly altered by elevated mucin and DNA content. Reduction in sputum pore size is characteristic of patients with obstructed airways as indicated by measured pulmonary function tests. Our microstructural read-out may serve as a novel biomarker for CF.

Fungal infections and treatment in cystic fibrosis.

PubMed

Middleton, Peter G; Chen, Sharon C-A; Meyer, Wieland

2013-11-01

This review summarizes some of the important recent findings concerning fungal airway infections in patients with cystic fibrosis (CF). For many years, both researchers and clinicians have focused on the problems in CF caused by chronic bacterial airway infection with organisms such as Haemophilus, Staphylococcus and Pseudomonas. However, until recently, the lack of sensitive culture techniques to isolate fungi in sputum, bronchoalveolar lavage fluid and other respiratory tract samples has limited the recognition of fungal species and their possible role in CF airway infections. Recent studies using fungal-selective culture media and molecular techniques have shown a plethora of different fungal species in the sputum expectorated from CF patients. Cross-sectional studies have shown associations between Aspergillus and Candida in the sputum and worse lung function. The presence of allergic bronchopulmonary aspergillosis is likely to be a negative prognostic factor, but whether simple fungal colonization itself indicates future problems is not clear. Current research is now examining these epidemiological associations to try to determine the clinical implications. This will help determine whether fungal colonization/infection is associated with worse outcome in CF patients. At present, there is no conclusive evidence that fungal organisms cause respiratory decline. Recent studies of antifungal therapy in CF patients have reported differing results, so further investigations in this area are needed.

Effect of Media Modified To Mimic Cystic Fibrosis Sputum on the Susceptibility of Aspergillus fumigatus, and the Frequency of Resistance at One Center

PubMed Central

Moss, Richard B.; Hernandez, Cathy; Clemons, Karl V.; Martinez, Marife

2016-01-01

Studies of cystic fibrosis (CF) patient exacerbations attributed to Pseudomonas aeruginosa infection have indicated a lack of correlation of outcome with in vitro susceptibility results. One explanation is that the media used for testing do not mimic the airway milieu, resulting in incorrect conclusions. Therefore, media have been devised to mimic CF sputum. Aspergillus fumigatus is the leading fungal pathogen in CF, and susceptibility testing is also used to decide therapeutic choices. We assessed whether media designed to mimic CF sputa would give different fungal susceptibility results than those of classical methods, assaying voriconazole, the most utilized anti-Aspergillus drug in this setting, and 30 CF Aspergillus isolates. The frequency of marked resistance (defined as an MIC of >4 μg/ml) in our CF unit by classical methods is 7%. Studies performed with classical methods and with digested sputum medium, synthetic sputum medium, and artificial sputum medium revealed prominent differences in Aspergillus susceptibility results, as well as growth rate, with each medium. Clinical correlative studies are required to determine which results are most useful in predicting outcome. Comparison of MICs with non-CF isolates also indicated the CF isolates were generally more resistant. PMID:26810647

Effect of Media Modified To Mimic Cystic Fibrosis Sputum on the Susceptibility of Aspergillus fumigatus, and the Frequency of Resistance at One Center.

PubMed

Stevens, David A; Moss, Richard B; Hernandez, Cathy; Clemons, Karl V; Martinez, Marife

2016-04-01

Studies of cystic fibrosis (CF) patient exacerbations attributed toPseudomonas aeruginosainfection have indicated a lack of correlation of outcome within vitrosusceptibility results. One explanation is that the media used for testing do not mimic the airway milieu, resulting in incorrect conclusions. Therefore, media have been devised to mimic CF sputum.Aspergillus fumigatusis the leading fungal pathogen in CF, and susceptibility testing is also used to decide therapeutic choices. We assessed whether media designed to mimic CF sputa would give different fungal susceptibility results than those of classical methods, assaying voriconazole, the most utilized anti-Aspergillusdrug in this setting, and 30 CFAspergillusisolates. The frequency of marked resistance (defined as an MIC of >4 μg/ml) in our CF unit by classical methods is 7%. Studies performed with classical methods and with digested sputum medium, synthetic sputum medium, and artificial sputum medium revealed prominent differences inAspergillussusceptibility results, as well as growth rate, with each medium. Clinical correlative studies are required to determine which results are most useful in predicting outcome. Comparison of MICs with non-CF isolates also indicated the CF isolates were generally more resistant. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Proteolytic cleavage of the long pentraxin PTX3 in the airways of cystic fibrosis patients.

PubMed

Hamon, Yveline; Jaillon, Sébastien; Person, Christine; Giniès, Jean-Louis; Garo, Erwan; Bottazzi, Barbara; Ghamrawi, Sarah; Urban, Thierry; Subra, Jean-François; Bouchara, Jean-Philippe; Mantovani, Alberto; Jeannin, Pascale; Delneste, Yves

2013-12-01

The prototypic long pentraxin PTX3, a soluble pattern recognition receptor, plays an important role in innate defense against selected pathogens by favoring their elimination and the initiation of protective responses. PTX3 has notably beneficial effects in mice infected with Aspergillus fumigatus and Pseudomonas aeruginosa. Cystic fibrosis (CF), a severe inherited autosomal recessive disease, is characterized by recurrent lung infections, especially by these two pathogens. We thus hypothesized that the status of PTX3 may be altered in CF patients. Level and integrity of PTX3 were analyzed in the sputum samples from 51 CF patients and 7 patients with chronic obstructive pulmonary disease (COPD). The levels of PTX3 were increased in serums from CF patients, but low in their respiratory secretions. PTX3 concentrations in sputum samples were dramatically lower in CF patients than in COPD patients. The low concentration of PTX3 resulted from a proteolysis cleavage by elastase and A. fumigatus proteases. Interestingly, the N-ter domain of PTX3, involved in protection against A. fumigatus, is preferentially degraded by these proteases. These results indicate that the selective proteolysis of PTX3 in the CF lung may explain, in part, the recurrent lung infections by PTX3-sensitive pathogens in CF patients.

Levofloxacin Pharmacokinetics in Adult Cystic Fibrosis

PubMed Central

Lee, Carlton K. K.; Boyle, Michael P.; Diener-West, Marie; Brass-Ernst, Lois; Noschese, Michelle; Zeitlin, Pamela L.

2007-01-01

Background Cystic fibrosis (CF) patients have enhanced renal clearance of aminoglycosides and several β-lactams and require higher dosages. Levofloxacin is a fluoroquinolone with extensive renal elimination and enhanced penetration into lungs and Pseudomonas aeruginosa (PA) biofilms. We studied the preliminary pharmacokinetic and pharmacodynamic (PK/PD) relationship of levofloxacin in CF. Methods Twelve patients at least 18 years old with a mild-to-moderate pulmonary exacerbation and fluoroquinolone-sensitive PA colonization received oral levofloxacin, 500 mg qd, for 14 days. Steady-state serum concentrations were collected after 3 to 7 days, and sputum samples for PA densities were collected before and after levofloxacin. PK/PD relationships for reducing PA sputum densities were evaluated. Results When compared to published data on non-CF patients, CF patients had similar area under the curve for 24 h (AUC24), total clearance, volume of distribution, maximum serum concentration (Cpmax), and elimination half-life: mean, 7.33 μg × h/mL/kg (SD, 1.70); 2.43 mL/min/kg (SD, 0.74); 1.33 L/kg (SD, 0.37); 7.06 μg/mL (SD, 2.35); and 6.44 h (SD, 1.1), respectively. Time to reach maximum serum concentration (Tmax) in CF was longer: mean, 2.20 h (SD, 0.99) vs 1.1 h (SD, 0.4) [p < 0.01]. Preliminary PK/PD analysis failed to demonstrate trends for decreasing PA sputum densities with increasing Cpmax/minimum inhibitory concentration (MIC) ratio and AUC24/MIC ratio. Conclusion CF levofloxacin pharmacokinetics corrected for body weight are similar to non-CF, except for Tmax. Standard levofloxacin dosing (especially monotherapy) is unlikely to produce maximum therapeutic effectiveness. Additional levofloxacin studies in CF are necessary to evaluate its sputum concentrations; the benefits of higher daily dosages (≥ 750 mg); and establish PK/PD targets for managing PA pulmonary infections. PMID:17356095

Detection of Bacteriophage Particles Containing Antibiotic Resistance Genes in the Sputum of Cystic Fibrosis Patients.

PubMed

Brown-Jaque, Maryury; Rodriguez Oyarzun, Lirain; Cornejo-Sánchez, Thais; Martín-Gómez, Maria T; Gartner, Silvia; de Gracia, Javier; Rovira, Sandra; Alvarez, Antonio; Jofre, Joan; González-López, Juan J; Muniesa, Maite

2018-01-01

Cystic fibrosis (CF) is a chronic disease in which the bacterial colonization of the lung is linked to an excessive inflammatory response that leads to respiratory failure. The microbiology of CF is complex. Staphylococcus aureus is the first bacterium to colonize the lungs in 30% of pediatric CF patients, and 80% of adult patients develop a chronic Pseudomonas aeruginosa infection, but other microorganisms can also be found. The use of antibiotics is essential to treat the disease, but antibiotic performance is compromised by resistance mechanisms. Among various mechanisms of transfer of antibiotic resistance genes (ARGs), the recently been reported bacteriophages are the least explored in clinical settings. To determine the role of phages in CF as mobile genetic elements (MGEs) carrying ARGs, we evaluated their presence in 71 CF patients. 71 sputum samples taken from these patients were screened for eight ARGs ( bla TEM , bla CTX-M-1 -group, bla CTX-M-9 -group, bla OXA-48 , bla VIM , mecA , qnrA , and qnrS ) in the bacteriophage DNA fraction. The phages found were also purified and observed by electron microscopy. 32.4% of CF patients harbored ARGs in phage DNA. β-lactamase genes, particularly bla VIM and bla TEM , were the most prevalent and abundant, whereas mecA , qnrA , and qnrS were very rare. Siphoviridae phage particles capable of infecting P. aeruginosa and Klebsiella pneumoniae were detected in CF sputum. Phage particles harboring ARGs were found to be abundant in the lungs of both CF patients and healthy individuals and could contribute to the colonization of multiresistant strains.

Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection.

PubMed

Kolpen, Mette; Kühl, Michael; Bjarnsholt, Thomas; Moser, Claus; Hansen, Christine Rønne; Liengaard, Lars; Kharazmi, Arsalan; Pressler, Tanja; Høiby, Niels; Jensen, Peter Østrup

2014-01-01

Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO3(-) and NO2(-) in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N2O (range 1.4-157.9 µM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3(-) decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.

Pharmacokinetics and Tolerability of Oral Sildenafil in Adults with Cystic Fibrosis Lung Disease

PubMed Central

Taylor-Cousar, JL; Wiley, C; Felton, LA; St Clair, C; Jones, M; Curran-Everett, D; Poch, K; Nichols, DP; Solomon, GM; Saavedra, MT; Accurso, FJ; Nick, JA

2014-01-01

Rationale Airway inflammation is central to cystic fibrosis (CF) pathophysiology. Pre-clinical models have shown that phosphodiesterase inhibitors (PDEi) like sildenafil have anti-inflammatory activity. PDEi have not been studied in CF subjects. Objectives We evaluated the pharmacokinetics, tolerability, and safety of sildenafil in subjects with CF. Sputum biomarkers were used to explore efficacy. Methods An open-label pilot study of oral sildenafil administration was conducted in adults with mild to moderate CF lung disease. Subjects received oral sildenafil 20 or 40 mg p.o. t.i.d. for 6 weeks. Measurements and Main Results Twenty subjects completed the study. Estimated elimination rate constants were statistically different in subjects with CF compared to previously published non-CF subjects. Side effects were generally mild. There were no drug-related serious adverse events. Sputum neutrophil elastase activity decreased. Conclusions Subjects with CF may eliminate sildenafil at a faster rate than non-CF subjects. Sildenafil administration was safe in subjects with CF, and decreased sputum elastase activity. Sildenafil warrants further study as an anti-inflammatory in CF. PMID:25466700

The many ways sputum flows - Dealing with high within-subject variability in cystic fibrosis sputum rheology.

PubMed

Radtke, Thomas; Böni, Lukas; Bohnacker, Peter; Fischer, Peter; Benden, Christian; Dressel, Holger

2018-04-21

We evaluated test-retest reliability of sputum viscoelastic properties in clinically stable patients with cystic fibrosis (CF). Data from a prospective, randomized crossover study was used to determine within-subject variability of sputum viscoelasticity (G', storage modulus and G", loss modulus at 1 and 10 rad s -1 ) and solids content over three consecutive visits. Precision of sputum properties was quantified by within-subject standard deviation (SD ws ), coefficient of variation (CV) and intraclass correlation coefficients (ICC). Fifteen clinically stable adults with CF (FEV 1 range 24-94% predicted) were included. No differences between study visits (mean ± SD 8 ± 2 days) were observed for any sputum rheology measure. CV's for G', G" and solids content ranged between 40.3-45.3% and ICC's between 0.21-0.42 indicating poor to fair test-retest reliability. Short-term within-subject variability of sputum properties is high in clinically stable adults with CF. Investigators applying shear rheology experiments in future prospective studies should consider using multiple measurements aiming to increase precision of sputum rheological outcomes. Copyright © 2018 Elsevier B.V. All rights reserved.

The CF-Sputum Induction Trial (CF-SpIT) to assess lower airway bacterial sampling in young children with cystic fibrosis: a prospective internally controlled interventional trial.

PubMed

Ronchetti, Katherine; Tame, Jo-Dee; Paisey, Christopher; Thia, Lena P; Doull, Iolo; Howe, Robin; Mahenthiralingam, Eshwar; Forton, Julian T

2018-06-01

Pathogen surveillance is challenging but crucial in children with cystic fibrosis-who are often non-productive of sputum even if actively coughing-because infection and lung disease begin early in life. The role of sputum induction as a diagnostic tool for infection has not previously been systematically addressed in young children with cystic fibrosis. We aimed to assess the pathogen yield from sputum induction compared with that from cough swab and single-lobe, two-lobe, and six-lobe bronchoalveolar lavage. This prospective internally controlled interventional trial was done at the Children's Hospital for Wales (Cardiff, UK) in children with cystic fibrosis aged between 6 months and 18 years. Samples from cough swab, sputum induction, and single-lobe, two-lobe, and six-lobe bronchoalveolar lavage were matched for within-patient comparisons. Primary outcomes were comparative pathogen yield between sputum induction and cough swab for stage 1, and between sputum induction, and single-lobe, two-lobe, and six-lobe bronchoalveolar lavage for stage 2. Data were analysed as per protocol. This study is registered with the UK Clinical Research Network (14615) and with the International Standard Randomised Controlled Trial Network Registry (12473810). Between Jan 23, 2012, and July 4, 2017, 124 patients were prospectively recruited to the trial and had 200 sputum induction procedures for stage 1. 167 (84%) procedures were successful and the procedure was well tolerated. Of the 167 paired samples, 63 (38%) sputum-induction samples were pathogen positive compared with 24 (14%) cough swabs (p<0·0001; odds ratio [OR] 7·5; 95% CI 3·19-17·98). More pathogens were isolated from sputum induction than cough swab (79 [92%] of 86 vs 27 [31%] of 86; p<0·0001). For stage 2, 35 patients had a total of 41 paired sputum-induction and bronchoalveolar lavage procedures. Of the 41 paired samples, 28 (68%) were positive for at least one of the concurrent samples. 39 pathogens were isolated. Sputum induction identified 27 (69%) of the 39 pathogens, compared with 22 (56%; p=0·092; OR 3·3, 95% CI 0·91-12·11) on single-lobe, 28 (72%; p=1·0; OR 1·1, 95% CI 0·41-3·15) on two-lobe, and 33 (85%; p=0·21; OR 2·2, 95% CI 0·76-6·33) on six-lobe bronchoalveolar lavage. Sputum induction is superior to cough swab for pathogen detection, is effective at sampling the lower airway, and is a credible surrogate for bronchoalveolar lavage in symptomatic children. A substantial number of bronchoscopies could be avoided if sputum induction is done first and pathogens are appropriately treated. Both sputum induction and six-lobe bronchoalveolar lavage provide independent, sizeable gains in pathogen detection compared with the current gold-standard two-lobe bronchoalveolar lavage. We propose that sputum induction and six-lobe bronchoalveolar lavage combined are used as standard of care for comprehensive lower airway pathogen detection in children with cystic fibrosis. Health and Care Research Wales-Academic Health Science Collaboration and Wellcome Trust Institutional Strategic Support Fund. Copyright © 2018 The Author(s). Published by Elsevier Ltd. Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

PubMed

Nunvar, Jaroslav; Kalferstova, Lucie; Bloodworth, Ruhi A M; Kolar, Michal; Degrossi, Jose; Lubovich, Silvina; Cardona, Silvia T; Drevinek, Pavel

2016-01-01

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

Intravenous antibiotics reduce the presence of Aspergillus in adult cystic fibrosis sputum.

PubMed

Baxter, Caroline G; Rautemaa, Riina; Jones, Andrew M; Webb, A Kevin; Bull, Matthew; Mahenthiralingam, Eshwar; Denning, David W

2013-07-01

Pseudomonas aeruginosa and Aspergillus fumigatus frequently co-colonise the airways of patients with cystic fibrosis (CF). This study aimed to assess the impact of short-term administration of intravenous antipseudomonal antibiotics during CF exacerbations on the presence of Aspergillus. Pre- and post-antibiotic sputum samples from 26 adult patients with CF and chronic Pseudomonas colonisation were analysed for the presence of Aspergillus by fungal culture, real-time PCR and galactomannan antigen (GM). Lung function (forced expiratory volume in 1 s and forced vital capacity % predicted) and blood levels of total IgE, specific A fumigatus IgE and specific A fumigatus IgG were measured at the start and end of antibiotics. Respiratory viral real-time PCR and bacterial community profiling using ribosomal intergenic spacer analysis (RISA) were performed to estimate concurrent changes in the lung microbiome. Aspergillus PCR and GM were more sensitive than culture in detecting Aspergillus species (culture 8%, GM 31%, PCR 77%). There was a significant decline in the presence of Aspergillus, measured both by PCR and GM index, following antibacterial therapy (PCR: median increase in crossing threshold 1.7 (IQR 0.5-3.8), p<0.001; GM: median fall in GM index 0.7 (IQR 0.4-1.6), p=0.016). All patients improved clinically with a significant increase in lung function (p<0.0001). RISA community analysis showed large changes in bacterial community similarity in 67% of patients following antibiotics. Viral RT-PCR demonstrated the presence of a concurrent respiratory virus in 27% of patients. Intravenous antibiotics targeting Pseudomonas during CF pulmonary exacerbations have a negative impact on the presence of Aspergillus in sputum samples.

Polyelectrolyte Structure and Interactions in Model Cystic Fibrosis Sputum

NASA Astrophysics Data System (ADS)

Slimmer, Scott; Angelini, Thomas; Liang, Hongjun; Butler, John; Wong, Gerard C. L.

2002-03-01

Cystic fibrosis sputum is a complex fluid consisting of a number of components, including mucin (a glycoprotein), lysozyme (a cationic polypeptide), water, salt, as well as a high concentration of a number of anionic biological polyelectrolytes such as DNA and F-actin. The interactions governing these components are poorly understood, but may have important clinical consequences. For example, the formation of these biological polyelectrolytes into ordered gel phases may contribute significantly to the observed high viscosity of CF sputum. In this work, a number of model systems were created to simulate CF sputum in vitro, in order to elucidate the contributions of the different components. Preliminary results will be presented. This work was supported by NSF DMR-0071761, DOE DEFG02-91ER45439, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

Actin - Lysozyme Interactions in Model Cystic Fibrosis Sputum

NASA Astrophysics Data System (ADS)

Sanders, Lori; Slimmer, Scott; Angelini, Thomas; Wong, Gerard C. L.

2003-03-01

Cystic fibrosis sputum is a complex fluid consisting of mucin (a glycoprotein), lysozyme (a cationic polypeptide), water, salt, as well as a high concentration of a number of anionic biological polyelectrolytes such as DNA and F-actin. The interactions governing these components are poorly understood, but may have important clinical consequences. For example, the formation of these biological polyelectrolytes into ordered gel phases may contribute significantly to the observed high viscosity of CF sputum. In this work, a number of model systems containing actin, lysozyme, and KCl were created to simulate CF sputum in vitro. These model systems were studied using small angle x-ray scattering and confocal fluorescence microscopy. Preliminary results will be presented. This work was supported by NSF DMR-0071761, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

Chronic Aspergillus fumigatus colonization of the pediatric cystic fibrosis airway is common and may be associated with a more rapid decline in lung function.

PubMed

Saunders, Rosalind V; Modha, Deborah E; Claydon, Alison; Gaillard, Erol A

2016-07-01

Filamentous fungi are commonly isolated from the respiratory tract of CF patients, but their clinical significance is uncertain and the reported incidence variable. We report on the degree of Aspergillus fumigatus airway colonization in a tertiary pediatric CF cohort, evaluate the sensitivity of routine clinical sampling at detecting A. fumigatus, and compare lung function of A. fumigatus-colonized and non-colonized children.We carried out an 8-year retrospective cohort analysis using local databases, examining 1024 respiratory microbiological specimens from 45 children. Nineteen (42%) had a positive A. fumigatus culture at least once during the 8-year period, with 10 (22%) children persistently colonized. Overall, 29% of 48 bronchoalveolar lavage (BAL) samples tested positive for A. fumigatus, compared with 14% of 976 sputum samples. Of 33 children for whom lung function data were available during the study period, seven were classed as having severe lung disease, of whom four (57%) were persistently colonized with A. fumigatus.We conclude that chronic A. fumigatus colonization of the CF airway is common, and may be associated with worse lung function. In our practice, BAL appears superior at detecting lower airway A. fumigatus compared to sputum samples. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pharmacokinetics and Safety of MP-376 (Levofloxacin Inhalation Solution) in Cystic Fibrosis Subjects▿

PubMed Central

Geller, David E.; Flume, Patrick A.; Griffith, David C.; Morgan, Elizabeth; White, Dan; Loutit, Jeffery S.; Dudley, Michael N.

2011-01-01

The pharmacokinetics and tolerability of nebulized MP-376 (levofloxacin inhalation solution [Aeroquin]) were determined in cystic fibrosis (CF) subjects. Ten CF subjects received single 180-mg doses of two formulations of MP-376, followed by a multiple-dose phase of 240 mg once daily for 7 days. Serum and expectorated-sputum samples were assayed for levofloxacin content. Safety was evaluated following the single- and multiple-dose study phases. Nebulized MP-376 produced high concentrations of levofloxacin in sputum. The mean maximum plasma concentration (Cmax) ranged between 2,563 and 2,932 mg/liter for 180-mg doses of the 50- and 100-mg/ml formulations, respectively. After 7 days of dosing, the mean Cmax for the 240-mg dose was 4,691 mg/liter. The mean serum levofloxacin Cmax ranged between 0.95 and 1.28 for the 180-mg doses and was 1.71 for the 240-mg dose. MP-376 was well tolerated. Nebulized MP-376 produces high sputum and low serum levofloxacin concentrations. The pharmacokinetics, safety, and tolerability were similar for the two formulations. MP-376 240 mg (100 mg/ml) is being advanced into late-stage clinical development. PMID:21444699

Aspergillus Bronchitis in Patients with Cystic Fibrosis.

PubMed

Brandt, Claudia; Roehmel, Jobst; Rickerts, Volker; Melichar, Volker; Niemann, Nadja; Schwarz, Carsten

2018-02-01

Aspergillus fumigatus frequently colonizes the airways of patients with cystic fibrosis (CF) and may cause various severe infections, such as bronchitis. Serological data, sputum dependent markers and longitudinal data of treated cases of Aspergillus bronchitis were evaluated for further description of this infection. This study, which comprises three substudies, aimed to analyze epidemiological data of Aspergillus in CF and the entity of Aspergillus bronchitis. In a first step, data of the German Cystic Fibrosis Registry were used to evaluate the frequency of Aspergillus colonization in patients with CF (n = 2599). Then a retrospective analysis of 10 cases of Aspergillus bronchitis was performed to evaluate longitudinal data for lung function and clinical presentation parameters: sputum production, cough and physical capacity. Finally, a prospective cohort study (n = 22) was conducted to investigate serological markers for Aspergillus bronchitis: total serum IgE, specific serum IgE, specific serum IgG, as well as sputum galactomannan, real-time PCR detection of Aspergillus DNA in sputum and fungal cultures. Analysis of the German CF registry revealed an Aspergillus colonization rate of 32.5% among the 2599 patients. A retrospective data analysis of 10 treated cases revealed the clinical course of Aspergillus bronchitis, including repeated positive sputum culture findings for A. fumigatus, no antibiotic treatment response, total serum IgE levels <200 kU/l, no observation of new pulmonary infiltrates and appropriate antifungal treatment response. Antifungal treatment durations of 4 ± 1.6 (2-6) weeks significantly reduced cough (P = 0.0067), sputum production (P < 0.0001) and lung function measures (P = 0.0358) but not physical capacity (P = 0.0794). From this retrospective study, a prevalence of 1.6% was calculated. In addition, two cases of Aspergillus bronchitis were identified in the prospective cohort study according to immunological, molecular and microbiological parameters. A prevalence of 9% was assessed. Aspergillus bronchitis appears to occur in a minority of colonized CF patients. Antifungal treatment may reduce respiratory symptoms and restore lung function.

Aspergillus/allergic bronchopulmonary aspergillosis in an Irish cystic fibrosis population: a diagnostically challenging entity.

PubMed

Chotirmall, Sanjay Haresh; Branagan, Peter; Gunaratnam, Cedric; McElvaney, Noel Gerard

2008-08-01

Patients with cystic fibrosis (CF) can become colonized by aspergillus, which can act as an allergen and cause allergic bronchopulmonary aspergillosis (ABPA). To determine the rate of aspergillus colonization and ABPA in a population of Irish patients with CF. In 50 consecutive patients with CF who presented with exacerbations, we looked for the presence of aspergillus in their sputum and signs and symptoms of ABPA. Fifteen patients (30%) grew aspergillus species in their sputum cultures. Six patients (12%) had ABPA. Matched for age, sex, genotype, and microbiology, there was no significant difference in forced expiratory volume in the first second (percent predicted, FEV(1)%) in subjects with aspergillus-positive sputum compared to those not colonized with aspergillus. Subjects with ABPA experienced sharp short-term deterioration in lung function (mean 6.7% predicted FEV(1)), which returned to baseline following at least 4 weeks of treatment. The prevalence of ABPA was 12%. Aspergillus-positive sputum of itself was not a poor prognostic sign in terms of lung function over the 5-year study course. ABPA produces short-term reversible declines in lung function and responds to treatment. The frequency of aspergillus isolates did not correlate with the occurrence of ABPA. A low threshold for the diagnosis of ABPA should be maintained in any patient with CF who does not improve with antibiotics.

Pharmacokinetics of Tedizolid in Plasma and Sputum of Adults with Cystic Fibrosis.

PubMed

Park, A Young J; Wang, Joshua; Jayne, Jordanna; Fukushima, Lynn; Rao, Adupa P; D'Argenio, David Z; Beringer, Paul M

2018-06-18

Over the past decade, the prevalence of infections involving Methicillin-resistant Staphylococcus aureus (MRSA) in patients with cystic fibrosis (CF) has increased significantly. Tedizolid (TZD) demonstrates excellent activity against MRSA and a favorable safety profile. The pharmacokinetics of several antibiotics has shown to be altered in CF patients. The purpose of this study was to characterize the pharmacokinetics of tedizolid in this population. Eleven patients with CF were randomized to receive tedizolid phosphate 200 mg PO or IV once daily for 3 doses, with minimum 2-day washout, followed by crossover to the remaining dosage form. Plasma and expectorated sputum were collected following the third dose of each dosage form for analysis. Population pharmacokinetics was performed using maximum-likelihood, expectation maximization method, and the disposition of TZD was described by a 2-compartment model. The sputum concentrations exceeded the unbound plasma concentrations with an estimated mean (%CV) sputum-to-unbound plasma penetration ratio of 2.88 (50.3). The estimated population mean ± standard deviation of total clearance, central volume of distribution, and bioavailability were 9.72 ± 1.62 L/h, 61.6 ± 6.94 L, and 1.04 ± 0.232 respectively. The total clearance is higher in CF patients when compared with healthy volunteers; however, it is similar to published data in patients with complicated skin and skin structure infections (cSSSI). This study demonstrates the oral bioavailability of tedizolid is excellent in patients with CF, and the plasma pharmacokinetics are similar to that reported for patients with cSSSI. Copyright © 2018 American Society for Microbiology.

Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure

PubMed Central

Henderson, Ashley G.; Ehre, Camille; Button, Brian; Abdullah, Lubna H.; Cai, Li-Heng; Leigh, Margaret W.; DeMaria, Genevieve C.; Matsui, Hiro; Donaldson, Scott H.; Davis, C. William; Sheehan, John K.; Boucher, Richard C.; Kesimer, Mehmet

2014-01-01

The pathogenesis of mucoinfective lung disease in cystic fibrosis (CF) patients likely involves poor mucus clearance. A recent model of mucus clearance predicts that mucus flow depends on the relative mucin concentration of the mucus layer compared with that of the periciliary layer; however, mucin concentrations have been difficult to measure in CF secretions. Here, we have shown that the concentration of mucin in CF sputum is low when measured by immunologically based techniques, and mass spectrometric analyses of CF mucins revealed mucin cleavage at antibody recognition sites. Using physical size exclusion chromatography/differential refractometry (SEC/dRI) techniques, we determined that mucin concentrations in CF secretions were higher than those in normal secretions. Measurements of partial osmotic pressures revealed that the partial osmotic pressure of CF sputum and the retained mucus in excised CF lungs were substantially greater than the partial osmotic pressure of normal secretions. Our data reveal that mucin concentration cannot be accurately measured immunologically in proteolytically active CF secretions; mucins are hyperconcentrated in CF secretions; and CF secretion osmotic pressures predict mucus layer–dependent osmotic compression of the periciliary liquid layer in CF lungs. Consequently, mucin hypersecretion likely produces mucus stasis, which contributes to key infectious and inflammatory components of CF lung disease. PMID:24892808

Sputum rheology changes in cystic fibrosis lung disease following two different types of physiotherapy: flutter vs autogenic drainage.

PubMed

App, E M; Kieselmann, R; Reinhardt, D; Lindemann, H; Dasgupta, B; King, M; Brand, P

1998-07-01

The aim of the present study was to investigate the efficacy of two frequently used physiotherapies (PTs) for the removal of bronchial secretions in cystic fibrosis (CF) lung disease: autogenic drainage (AD) and the Flutter (Desitin in Germany). AD is believed to improve mucus clearance from peripheral to central airways due to airway caliber changes in combination with a special breathing technique. The Flutter is an easy-to-use physiotherapy device based on oscillations of a steel ball during expiration through a pipe-type device. To evaluate the acute and chronic physiotherapy effects of these two techniques, 14 CF patients underwent either twice daily AD or Flutter treatment for 4 consecutive weeks in a randomized crossover design. Prior to each therapy interval, for a 1-week wash-out period, no PT was administered, but patients continued regular medication. At the beginning and end of each 4-week interval, pulmonary function was measured before and after an acute 30-min therapy. At the end of the PT session, sputum was collected, weighed, and deep frozen until analyzed. The viscoelasticity of the sputum was evaluated using a magnetic microrheometer. No significant changes were noted for FVC, FEV1, or sputum volume throughout the study. Sputum viscoelasticity (rigidity index), however, was significantly lower (p<0.01) after therapy with the Flutter in comparison with AD, predicting improvements in mucociliary and cough clearability of the secretions. In a companion in vitro experiment, oscillations generated by passing humidified air over CF sputum lining an acrylic tube connected to a Flutter de-ice were found to decrease sputum elasticity, as measured by a filancemeter. These findings suggest that applied oscillations are capable of decreasing mucus viscoelasticity within the airways at frequencies and amplitudes achievable with the Flutter device, and provide direct evidence that PT can reduce the viscoelasticity of sputum.

Is bronchoscopy an obsolete tool in cystic fibrosis? The role of bronchoscopy in cystic fibrosis and its clinical use

PubMed Central

2017-01-01

Cystic fibrosis (CF) is a progressive life threatening multisystem genetic disease which affects the CF transmembrane conductance regulator channel. Respiratory causes remain the most common mortality in CF. With the onset of newborn screening, initiating treatments both for prophylaxis and disease management, optimizing nutritional support, and developing therapies targeting CF transmembrane conductance regulator protein, this has significantly changed the face of managing this devastating disease. Bronchoscopy and related procedures such as bronchoalveolar lavage (BAL), transbronchial biopsies, and protected brush sampling have been looked at in the management of CF as patients with CF continue to live longer with the help of newer therapies, the microbiome in the lung becomes less diverse along with increased occurrences for noninfectious causes of airway diseases. Though bronchoscopy has been used in conjunction with other modalities such as computed tomography and sputum induction providing a better understanding of the progression of the disease, it still remains valuable in the diagnosis and management of CF. PMID:29214071

Serum Iron Level Is Associated with Time to Antibiotics in Cystic Fibrosis.

PubMed

Gifford, Alex H; Dorman, Dana B; Moulton, Lisa A; Helm, Jennifer E; Griffin, Mary M; MacKenzie, Todd A

2015-12-01

Serum levels of hepcidin-25, a peptide hormone that reduces blood iron content, are elevated when patients with cystic fibrosis (CF) develop pulmonary exacerbation (PEx). Because hepcidin-25 is unavailable as a clinical laboratory test, we questioned whether a one-time serum iron level was associated with the subsequent number of days until PEx, as defined by the need to receive systemic antibiotics (ABX) for health deterioration. Clinical, biochemical, and microbiological parameters were simultaneously checked in 54 adults with CF. Charts were reviewed to determine when they first experienced a PEx after these parameters were assessed. Time to ABX was compared in subgroups with and without specific attributes. Multivariate linear regression was used to identify parameters that significantly explained variation in time to ABX. In univariate analyses, time to ABX was significantly shorter in subjects with Aspergillus-positive sputum cultures and CF-related diabetes. Multivariate linear regression models demonstrated that shorter time to ABX was associated with younger age, lower serum iron level, and Aspergillus sputum culture positivity. Serum iron, age, and Aspergillus sputum culture positivity are factors associated with shorter time to subsequent PEx in CF adults. © 2015 Wiley Periodicals, Inc.

Automated CT Scan Scores of Bronchiectasis and Air Trapping in Cystic Fibrosis

PubMed Central

Swiercz, Waldemar; Heltshe, Sonya L.; Anthony, Margaret M.; Szefler, Paul; Klein, Rebecca; Strain, John; Brody, Alan S.; Sagel, Scott D.

2014-01-01

Background: Computer analysis of high-resolution CT (HRCT) scans may improve the assessment of structural lung injury in children with cystic fibrosis (CF). The goal of this cross-sectional pilot study was to validate automated, observer-independent image analysis software to establish objective, simple criteria for bronchiectasis and air trapping. Methods: HRCT scans of the chest were performed in 35 children with CF and compared with scans from 12 disease control subjects. Automated image analysis software was developed to count visible airways on inspiratory images and to measure a low attenuation density (LAD) index on expiratory images. Among the children with CF, relationships among automated measures, Brody HRCT scanning scores, lung function, and sputum markers of inflammation were assessed. Results: The number of total, central, and peripheral airways on inspiratory images and LAD (%) on expiratory images were significantly higher in children with CF compared with control subjects. Among subjects with CF, peripheral airway counts correlated strongly with Brody bronchiectasis scores by two raters (r = 0.86, P < .0001; r = 0.91, P < .0001), correlated negatively with lung function, and were positively associated with sputum free neutrophil elastase activity. LAD (%) correlated with Brody air trapping scores (r = 0.83, P < .0001; r = 0.69, P < .0001) but did not correlate with lung function or sputum inflammatory markers. Conclusions: Quantitative airway counts and LAD (%) on HRCT scans appear to be useful surrogates for bronchiectasis and air trapping in children with CF. Our automated methodology provides objective quantitative measures of bronchiectasis and air trapping that may serve as end points in CF clinical trials. PMID:24114359

IgE-mediated immune responses and airway detection of Aspergillus and Candida in adult cystic fibrosis.

PubMed

Baxter, Caroline G; Moore, Caroline B; Jones, Andrew M; Webb, A Kevin; Denning, David W

2013-05-01

The recovery of Aspergillus and Candida from the respiratory secretions of patients with cystic fibrosis (CF) is common. Their relationship to the development of allergic sensitization and effect on lung function has not been established. Improved techniques to detect these organisms are needed to increase knowledge of these effects. A 2-year prospective observational cohort study was performed. Fifty-five adult patients with CF had sputum monitored for Aspergillus by culture and real-time polymerase chain reaction and Candida by CHROMagar and carbon assimilation profile (API/ID 32C). Skin prick tests and ImmunoCAP IgEs to a panel of common and fungal allergens were performed. Lung function and pulmonary exacerbation rates were monitored over 2 years. Sixty-nine percent of patient sputum samples showed chronic colonization with Candida and 60% showed colonization with Aspergillus. There was no association between the recovery of either organism and the presence of specific IgE responses. There was no difference in lung function decline for patients with Aspergillus or Candida colonization compared with those without (FEV₁ percent predicted, P = .41 and P = .90, respectively; FVC % predicted, P = .87 and P = .37, respectively). However, there was a significantly greater decline in FEV1 and increase in IV antibiotic days for those sensitized to Aspergillus (FEV₁ decline, P = .03; IV antibiotics days, P = .03). Allergic sensitization is not associated with recovery of Candida or Aspergillus from the sputum of patients with CF. Aspergillus but not Candida sensitization is associated with greater lung function decline and pulmonary exacerbations.

Secreted mucins and airway bacterial colonization in non-CF bronchiectasis.

PubMed

Sibila, Oriol; Suarez-Cuartin, Guillermo; Rodrigo-Troyano, Ana; Fardon, Thomas C; Finch, Simon; Mateus, Eder Freddy; Garcia-Bellmunt, Laia; Castillo, Diego; Vidal, Silvia; Sanchez-Reus, Ferran; Restrepo, Marcos I; Chalmers, James D

2015-10-01

Secreted mucins play a key role in antibacterial defence in the airway, but have not previously been characterized in non-cystic fibrosis (CF) bronchiectasis patients. We aim to investigate the relationship between secreted mucins levels and the presence of bacterial colonization due to potentially pathogenic microorganisms (PPM) in the airways of stable bronchiectasis patients. Clinically stable bronchiectasis patients were studied prospectively at two centres. Patients with other pulmonary conditions were excluded. Spontaneous sputum was subject to bacterial culture, and secreted mucins (MUC2, MUC5AC and MUC5B) were measured in sputum supernatants by ELISA. A total of 50 patients were included. PPM were identified from sputum samples in 30 (60%), with Pseudomonas aeruginosa (n = 10) and Haemophilus influenzae (n = 10) as the most common PPM. There were no baseline differences among airway colonized and non-colonized patients. Patients with airways colonized by PPM presented higher levels of airway MUC2. No differences in MUC5AC levels were found among groups, whereas MUC5B levels were undetectable. Patients with P. aeruginosa colonization expressed the highest levels of MUC2. High levels of MUC2 and MUC5AC are also correlated with disease severity using the Bronchiectasis Severity Index. Airway MUC2 levels were higher in bronchiectasis patients colonized with PPM compared with those without airway colonization, especially in patients with P. aeruginosa. These findings suggest that airway-secreted mucins levels may play a role in the pathogenesis of airway infection in non-CF bronchiectasis. © 2015 Asian Pacific Society of Respirology.

A novel chromogenic medium for isolation of Pseudomonas aeruginosa from the sputa of cystic fibrosis patients.

PubMed

Laine, Larissa; Perry, John D; Lee, Jenner; Oliver, Michelle; James, Arthur L; De La Foata, Corinne; Halimi, Diane; Orenga, Sylvain; Galloway, Angela; Gould, F Kate

2009-03-01

A novel chromogenic medium for isolation and identification of Pseudomonas aeruginosa from sputa of cystic fibrosis (CF) patients was evaluated and compared with standard laboratory methods. One hundred sputum samples from distinct CF patients were cultured onto blood agar (BA), Pseudomonas CN selective agar (CN) and a Pseudomonas chromogenic medium (PS-ID). All Gram-negative morphological variants from each medium were subjected to antimicrobial susceptibility testing, and identification using a combination of biochemical and molecular methods. P. aeruginosa was isolated from 62 samples after 72 h incubation. Blood agar recovered P. aeruginosa from 56 samples (90.3%) compared with 59 samples (95.2%) using either CN or PS-ID. The positive predictive value of PS-ID (98.3%) was significantly higher than growth on CN (88.5%) for identification of P. aeruginosa (P<0.05). PS-ID is a promising medium allowing for the isolation and simultaneous identification of P. aeruginosa from sputa of CF patients.

One time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.

PubMed

Boutin, Sébastien; Weitnauer, Michael; Hassel, Selina; Graeber, Simon Y; Stahl, Mirjam; Dittrich, A Susanne; Mall, Marcus A; Dalpke, Alexander H

2018-05-01

Chronic airway infection with Pseudomonas aeruginosa is a major risk factor of progression of lung disease in patients with cystic fibrosis (CF). Chronic P. aeruginosa infection evolves from intermittent infection that is amenable to antibiotic eradication, whereas chronically adapted P. aeruginosa becomes resistant to antibiotic therapy. Discrimination of intermittent versus chronic infection is therefore of high therapeutic relevance, yet the available diagnostic methods are only partly satisfactory. The aim of the present study was, therefore, to evaluate the usage of quantitative PCR (qPCR) to measure pathogen abundance and to discriminate between intermittent and chronic Pseudomonas infection in patients with CF. Using an established qPCR protocol, we analyzed the abundance of P. aeruginosa in 141 throats swabs and 238 sputa from CF patients with intermittent or chronic infection with P. aeruginosa, as determined by standard culture based diagnostics. We observed a large increase of abundance of P. aeruginosa in throat swabs and sputum samples from patients with chronic compared to intermittent infections with P. aeruginosa. The data show that abundance of P. aeruginosa as measured by qPCR is a valuable tool to discriminate intermittent from chronic infection. Of note, P. aeruginosa burden seems more sensitive than mucoidity phenotype to discriminate chronic from intermittent strains. Furthermore we observed that molecular detection in throat swabs was linked to a viable culture in the sputum when sputum was available. This result is of special interest in young patients with cystic fibrosis that often cannot expectorate sputum. We also observed that qPCR in comparison to culture detected the infection earlier. The results suggest that qPCR detection and quantification of P. aeruginosa is a precious tool to be added to the diagnostic toolbox in cystic fibrosis. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease.

PubMed

Sass, Laura A; Hair, Pamela S; Perkins, Amy M; Shah, Tushar A; Krishna, Neel K; Cunnion, Kenji M

2015-01-01

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (rs = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid.

Persistent oseltamivir-resistant pandemic influenza A/H1N1 infection in an adult with cystic fibrosis

PubMed Central

Flight, William George; Bright-Thomas, Rowland; Mutton, Kenneth; Webb, Kevin; Jones, Andrew

2011-01-01

The authors report the case of a 25-year-old patient with cystic fibrosis (CF) who developed pandemic influenza A/H1N1 during a visit to the USA in August 2010. The patient has severe CF lung disease and takes maintenance oral corticosteroids. The influenza virus was positive for the H275Y oseltamivir-resistance mutation despite the patient never having received oseltamivir. The patient has remained sputum-positive for over 4 months despite inhaled zanamivir therapy. This is the first reported case of transmission of oseltamivir-resistant H1N1 influenza to a patient with CF. The frequency of prolonged sputum carriage of pandemic influenza and transmission of oseltamivir-resistant strains are unknown on a population level. However, if our observations are replicated in other CF patients, they are potentially of considerable importance to clinical and infection-control practices in this patient group. PMID:22696672

A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

PubMed Central

2010-01-01

Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum. PMID:20637114

Smartphone-Based pH Sensor for Home Monitoring of Pulmonary Exacerbations in Cystic Fibrosis

PubMed Central

Sun, Alexander; Phelps, Tom; Yao, Chengyang; Venkatesh, A. G.; Conrad, Douglas; Hall, Drew A.

2017-01-01

Currently, Cystic Fibrosis (CF) patients lack the ability to track their lung health at home, relying instead on doctor checkups leading to delayed treatment and lung damage. By leveraging the ubiquity of the smartphone to lower costs and increase portability, a smartphone-based peripheral pH measurement device was designed to attach directly to the headphone port to harvest power and communicate with a smartphone application. This platform was tested using prepared pH buffers and sputum samples from CF patients. The system matches within ~0.03 pH of a benchtop pH meter while fully powering itself and communicating with a Samsung Galaxy S3 smartphone paired with either a glass or Iridium Oxide (IrOx) electrode. The IrOx electrodes were found to have 25% higher sensitivity than the glass probes at the expense of larger drift and matrix sensitivity that can be addressed with proper calibration. The smartphone-based platform has been demonstrated as a portable replacement for laboratory pH meters, and supports both highly robust glass probes and the sensitive and miniature IrOx electrodes with calibration. This tool can enable more frequent pH sputum tracking for CF patients to help detect the onset of pulmonary exacerbation to provide timely and appropriate treatment before serious damage occurs. PMID:28556804

Smartphone-Based pH Sensor for Home Monitoring of Pulmonary Exacerbations in Cystic Fibrosis.

PubMed

Sun, Alexander; Phelps, Tom; Yao, Chengyang; Venkatesh, A G; Conrad, Douglas; Hall, Drew A

2017-05-30

Currently, Cystic Fibrosis (CF) patients lack the ability to track their lung health at home, relying instead on doctor checkups leading to delayed treatment and lung damage. By leveraging the ubiquity of the smartphone to lower costs and increase portability, a smartphone-based peripheral pH measurement device was designed to attach directly to the headphone port to harvest power and communicate with a smartphone application. This platform was tested using prepared pH buffers and sputum samples from CF patients. The system matches within ~0.03 pH of a benchtop pH meter while fully powering itself and communicating with a Samsung Galaxy S3 smartphone paired with either a glass or Iridium Oxide (IrOx) electrode. The IrOx electrodes were found to have 25% higher sensitivity than the glass probes at the expense of larger drift and matrix sensitivity that can be addressed with proper calibration. The smartphone-based platform has been demonstrated as a portable replacement for laboratory pH meters, and supports both highly robust glass probes and the sensitive and miniature IrOx electrodes with calibration. This tool can enable more frequent pH sputum tracking for CF patients to help detect the onset of pulmonary exacerbation to provide timely and appropriate treatment before serious damage occurs.

Mucus-penetrating solid lipid nanoparticles for the treatment of cystic fibrosis: Proof of concept, challenges and pitfalls.

PubMed

Nafee, N; Forier, K; Braeckmans, K; Schneider, M

2018-03-01

Nanocarrier-mediated transmucosal drug delivery based on conventional mucoadhesive, muco-inert or mucus-penetrating nanoparticles (NPs) is a growing field especially in challenging diseases like cystic fibrosis (CF). Efficacy of such systems dictates profound investigation of particle-mucus interaction and factors governing the whole process. Although variable techniques studying particle diffusion in mucus have been introduced, standardized procedures are lacking. The study comprised different methods based on micro- and macro-displacement as well as colloidal stability and turbidimetric experiments. Artificial sputum medium (ASM), CF sputum and mucus-secreting cell line (Calu-3 air interface culture, AIC) were applied. Solid lipid nanoparticles (SLNs) coated with variable hydrophilic sheath (poloxamer, Tween 80 or PVA) represented the nanocarriers under investigation. Both micro-displacement studies based on single particle tracking and macro-displacement experiments based on 3D-time laps confocal imaging revealed faster diffusion of poloxamer- > Tween- > PVA-coated SLNs. Compared to ASM, CF sputum showed not only lower diffusion rates but also remarkable discrepancies in particle-mucus diffusion rate due to sputum heterogenicity. Meanwhile, in case of Calu-3 AIC, thickness of the mucosal layer as well as density of mucus network were key determinants in the diffusion process. The points emphasized in this study highlight the road towards in vivo relevant particle-mucus interaction research. Copyright © 2018 Elsevier B.V. All rights reserved.

Exhaled breath condensate adenosine tracks lung function changes in cystic fibrosis

PubMed Central

Olsen, Bonnie M.; Lin, Feng-Chang; Fine, Jason; Boucher, Richard C.

2013-01-01

Measurement of exhaled breath condensate (EBC) biomarkers offers a noninvasive means to assess airway disease, but the ability of EBC biomarkers to track longitudinal changes in disease severity remains unproven. EBC was collected from pediatric patients with cystic fibrosis (CF) during regular clinic visits over 1 yr. EBC biomarkers urea, adenosine (Ado), and phenylalanine (Phe) were measured by mass spectrometry, and biomarker ratios were used to control for variable dilution of airway secretions. EBC biomarker ratios were assessed relative to lung function in longitudinal, multivariate models and compared with sputum inflammatory markers and quality of life assessment (CFQ-R). EBC was successfully analyzed from 51 subjects during 184 visits (3.6 ± 0.9 visits per subject). EBC Ado/urea ratio was reproducible in duplicate samples (r = 0.62, P < 0.01, n = 20) and correlated with sputum neutrophil elastase (β = 2.5, P < 0.05). EBC Ado/urea correlated with the percentage predicted of forced expiratory volume in 1 s in longitudinal, multivariate models (β = −2.9, P < 0.01); EBC Ado/Phe performed similarly (β = −2.1, P < 0.05). In contrast, IL-8 and elastase measured in spontaneously expectorated sputum (n = 57 samples from 25 subjects) and the CFQ-R respiratory scale (n = 90 tests from 47 subjects) were not significantly correlated with lung function. EBC was readily collected in a clinic setting from a wide range of subjects. EBC Ado tracked longitudinal changes in lung function in CF, with results similar to or better than established measures. PMID:23355385

Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.

PubMed

Kennedy, Sarah; Beaudoin, Trevor; Yau, Yvonne C W; Caraher, Emma; Zlosnik, James E A; Speert, David P; LiPuma, John J; Tullis, Elizabeth; Waters, Valerie

2016-01-01

Pulmonary infection with Burkholderia cepacia complex in cystic fibrosis (CF) patients is associated with more-rapid lung function decline and earlier death than in CF patients without this infection. In this study, we used confocal microscopy to visualize the effects of various concentrations of tobramycin, achievable with systemic and aerosolized drug administration, on mature B. cepacia complex biofilms, both in the presence and absence of CF sputum. After 24 h of growth, biofilm thickness was significantly reduced by exposure to 2,000 μg/ml of tobramycin for Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis; 200 μg/ml of tobramycin was sufficient to reduce the thickness of Burkholderia dolosa biofilm. With a more mature 48-h biofilm, significant reductions in thickness were seen with tobramycin at concentrations of ≥100 μg/ml for all Burkholderia species. In addition, an increased ratio of dead to live cells was observed in comparison to control with tobramycin concentrations of ≥200 μg/ml for B. cepacia and B. dolosa (24 h) and ≥100 μg/ml for Burkholderia cenocepacia and B. dolosa (48 h). Although sputum significantly increased biofilm thickness, tobramycin concentrations of 1,000 μg/ml were still able to significantly reduce biofilm thickness of all B. cepacia complex species with the exception of B. vietnamiensis. In the presence of sputum, 1,000 μg/ml of tobramycin significantly increased the dead-to-live ratio only for B. multivorans compared to control. In summary, although killing is attenuated, high-dose tobramycin can effectively decrease the thickness of B. cepacia complex biofilms, even in the presence of sputum, suggesting a possible role as a suppressive therapy in CF. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Serum (1→3)-β-D-glucan and galactomannan levels in patients with cystic fibrosis: a retrospective cohort study.

PubMed

Träger, Johannes; Melichar, Volker Otto; Meyer, Renate; Rauh, Manfred; Bogdan, Christian; Held, Jürgen

2018-03-27

Aspergillus fumigatus is frequently encountered in sputum samples of patients with cystic fibrosis (CF), which traditionally has been interpreted as saprophytic airway colonization. However, this mere bystander role has been challenged by recent data. There is now evidence that Aspergillus fumigatus accelerates the decline of pulmonary function. (1→3)-β-D-glucan (BDG) and galactomannan (GM) are highly sensitive fungal biomarkers that are used to diagnose invasive fungal disease. However, their diagnostic value in CF patients is largely unknown. We conducted a retrospective cohort study on 104 CF patients to determine whether serum BDG and GM levels correlate with parameters such as Aspergillus-positive sputum cultures and lung function. Aspergillus fumigatus was persistently detected in 22 of the 104 CF patients (21%). Mean serum BDG and GM levels in the Aspergillus-positive patients were significantly higher than in those without persistent Aspergillus detection (89 versus 40 pg/ml [p = 0.022] and 0.30 versus 0.15 ODI [p = 0.013], respectively). 27 and 7 patients had elevated BDG (≥ 60 pg/ml) or GM levels (> 0.5 ODI), respectivly. BDG and GM levels showed a significant correlation (p = 0.004). Patients with increased serum concentrations of BDG were more frequently Aspergillus-positive (40.7 versus 14.3%, p = 0.004) and had a significantly lower forced expiratory volume in one second (FEV1) than patients with a normal BDG (61.6 versus 77.1%, p = 0.007). In the multivariate analysis, BDG but not GM or the growth of A. fumigatus, proved to be an independent predictor for the FEV1. CF patients with persistent Aspergillus detection have elevated BDG and GM levels which ranged between healthy and invasively infected patients. Serum BDG may be superior to GM and fungal culture in predicting an impaired lung function in CF patients.

Scedosporium apiospermum complex in cystic fibrosis; should we treat?

PubMed

Noni, Maria; Katelari, Anna; Kapi, Aikaterini; Stathi, Angeliki; Dimopoulos, George; Doudounakis, Stavros-Eleftherios

2017-09-01

Species of the Scedosporium apiospermum complex are the second most frequent filamentous fungi after Aspergillus fumigatus that can be found in cystic fibrosis (CF). Mixed colonisation by S. apiospermum complex and A. fumigatus is also quite common. In this study we summarise all CF patients who were colonised by S. apiospermum complex during their childhood and we present two CF patients who were treated as fungal bronchitis due to S. apiospermum complex. The medical records of 400 CF patients were reviewed in order to identify those with positive respiratory cultures for S. apiospermum complex. Scedosporium apiospermum complex was isolated in 10 CF patients and six of them had more than two positive sputum cultures during the study period. By the time of first isolation, the median age was 14.5 years, the median BMI was 19.41 kg/m 2 , the median predicted FEV 1 % was 78.65% and six patients had a history of A. fumigatus isolation. Two patients presented symptoms of infection while they were colonised by S. apiospermum complex. A rapid remission of their symptoms was observed only when antifungal therapy was administered. Antifungal treatment should be considered in CF patients who present symptoms of infection not responding to antibacterial therapy and S. apiospermum complex is persistently growing in sputum cultures. © 2017 Blackwell Verlag GmbH.

An Open-Label Investigation of the Pharmacokinetics and Tolerability of Oral Cysteamine in Adults with Cystic Fibrosis.

PubMed

Devereux, Graham; Steele, Sandra; Griffiths, Kairen; Devlin, Edward; Fraser-Pitt, Douglas; Cotton, Seonaidh; Norrie, John; Chrystyn, Henry; O'Neil, Deborah

2016-08-01

Cysteamine is licensed for use in nephropathic cystinosis but preclinical data suggest a role in managing cystic fibrosis (CF). This study aimed to determine whether oral cysteamine is absorbed in adult CF patients and enters the bronchial secretions. Tolerability outcomes were also explored. Patients ≥18 years of age, weighing >50 kg with stable CF lung disease were commenced on oral cysteamine bitartrate (Cystagon(®)) 450 mg once daily, increased weekly to 450 mg four times daily. Serial plasma cysteamine concentrations were measured for 24 h after the first dose. Participants were reviewed every week for 6 weeks, except at 4 weeks. Plasma cysteamine concentrations were measured 8 h after dosing when reviewed at 1, 2 and 3 weeks and 6 h after dosing when reviewed at 5 weeks. Sputum cysteamine concentration was also quantified at the 5-week assessment. Seven of the ten participants reported adverse reactions typical of cysteamine, two participants discontinued intervention. Following the first 450-mg dose, mean (SD) maximum concentration (C max) was 2.86 (1.96) mg/l, the time corresponding to C max (T max) was 1.2 (0.7) h, the half-life (t ½) was 3.7 (1.7) h, clearance (CL/F) 89.9 (30.5) L/h and volume of distribution (V d/F) 427 (129) L. Cysteamine appeared to accumulate in sputum with a median (interquartile range) sputum:plasma cysteamine concentration ratio of 4.2 (0.98-8.84). Oral cysteamine is absorbed and enters the bronchial secretions in patients with CF. Although adverse reactions were common, the majority of patients continued with cysteamine. Further trials are required to establish the risk benefit ratio of cysteamine therapy in CF.

Comparison of Ambient and Atmospheric Pressure Ion Sources for Cystic Fibrosis Exhaled Breath Condensate Ion Mobility-Mass Spectrometry Metabolomics

NASA Astrophysics Data System (ADS)

Zang, Xiaoling; Pérez, José J.; Jones, Christina M.; Monge, María Eugenia; McCarty, Nael A.; Stecenko, Arlene A.; Fernández, Facundo M.

2017-08-01

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The vast majority of the mortality is due to progressive lung disease. Targeted and untargeted CF breath metabolomics investigations via exhaled breath condensate (EBC) analyses have the potential to expose metabolic alterations associated with CF pathology and aid in assessing the effectiveness of CF therapies. Here, transmission-mode direct analysis in real time traveling wave ion mobility spectrometry time-of-flight mass spectrometry (TM-DART-TWIMS-TOF MS) was tested as a high-throughput alternative to conventional direct infusion (DI) electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) methods, and a critical comparison of the three ionization methods was conducted. EBC was chosen as the noninvasive surrogate for airway sampling over expectorated sputum as EBC can be collected in all CF subjects regardless of age and lung disease severity. When using pooled EBC collected from a healthy control, ESI detected the most metabolites, APCI a log order less, and TM-DART the least. TM-DART-TWIMS-TOF MS was used to profile metabolites in EBC samples from five healthy controls and four CF patients, finding that a panel of three discriminant EBC metabolites, some of which had been previously detected by other methods, differentiated these two classes with excellent cross-validated accuracy.

Photoacoustic analysis of the solubilization kinetics of pulmonary secretions from cystic fibrosis patients - secretor and non-secretor phenotypes

NASA Astrophysics Data System (ADS)

Barja, P. R.; Coelho, C. C.; Paiva, R. F.; Barboza, M. A.; Matos, L. C.; Matos, C. C. B.; Oliveira, L. V. F.

2010-03-01

Cystic fibrosis (CF) is an autosomal recessive inherited disease that increases viscoelasticity of pulmonary secretions. Affected patients are required to use therapeutic aerosols continuously. The expression of ABH glycoconjugates in exocrine secretions determines the nature of part of the carbohydrates present in these secretions, allowing the classification of individuals into the so-called "secretor" and "non secretor" phenotypes. The aim of this work was to employ photoacoustic (PA) measurements to monitor the solubilization kinetics of pulmonary secretions from CF patients, analyzing the influence of the secretor status in the solubilization kinetics of samples nebulized with different therapeutic aerosols. Sputum samples were obtained by spontaneous expectoration from positive and negative secretor CF patients. Each sample was nebulized with i) tobramycin, ii) alpha dornase, and iii) N-acetylcysteine in a PA cell; fitting of the data with the Boltzmann equation led to the determination of t0 (typical interaction time) and Δt (solubilization interval) for each curve. Differences between the secretor and non-secretor phenotypes were statistically significant in the groups for tobramycin and alpha dornase, but not for N-acetylcysteine. Results show that the secretor status influences the solubilization of pulmonary mucus of CF patients nebulized with tobramycin and alpha dornase.

Extensive cultivation of soil and water samples yields various pathogens in patients with cystic fibrosis but not Burkholderia multivorans.

PubMed

Peeters, Charlotte; Depoorter, Eliza; Praet, Jessy; Vandamme, Peter

2016-11-01

While the epidemiology of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients suggests that Burkholderia multivorans is acquired from environmental sources, this species has rarely been isolated from soil and water samples. Multiple isolation strategies were applied to water and soil samples that were previously shown to be B. multivorans PCR positive. These included direct plating and liquid enrichment procedures and the use of selective media, acclimatizing recovery and co-cultivation with CF sputum. MALDI-TOF mass spectrometry and sequence analysis of 16S rRNA and housekeeping genes were used to identify all isolates. None of the approaches yielded B. multivorans isolates. Other Burkholderia species, several Gram-negative non-fermenting bacteria (including Cupriavidus, Inquilinus, Pandoraea, Pseudomonas and Stenotrophomonas) and rapidly growing mycobacteria (including Mycobacterium chelonae) were all isolated from water and soil samples. The use of Bcc isolation media yielded a surprisingly wide array of rare but often clinically relevant CF pathogens, confirming that soil and water are reservoirs of these infectious agents. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ilowite, J.S.; Gorvoy, J.D.; Smaldone, G.C.

In cystic fibrosis (CF), the clinical effectiveness of aerosolized antibiotics is controversial. Previous investigators have not considered the type of nebulizer, droplet size, and dose to the lung in assessing the results of aerosol therapy. The present study tests the importance of these factors by standardizing an aerosol system for delivery of antibiotics and other agents to patients with CF. Particle size, distribution, and output from a commercially available nebulizer were measured. Thirteen patients with CF inhaled aerosol (MMAD = 1.1 micron) containing gentamicin (160 mg in nebulizer) and /sup 99m/Tc-labeled human serum albumin. Patients' sputum and serum were analyzedmore » for gentamicin levels by immunoenzymatic assay. Using a gamma camera and suitable filters, central versus peripheral deposition (C/P ratio) and whole lung deposition were measured and related to sputum gentamicin levels. Gentamicin deposit averaged 12.3 mg +/- 5.9 (SD) or 7.69% of the original amount placed in the nebulizer. Peak sputum levels averaged 376.6 micrograms/ml +/- 275, whereas serum levels were undetectable in all patients. When peak sputum levels were normalized for the amount deposited, a close correlation with C/P ratio was obtained (r = 0.88, p less than 0.05). Furthermore, an inverse relationship was found between the C/P ratio and the %FEV1 (r = 0.76, p less than 0.05). Finally, a bell-shaped relationship between deposited dose and minute ventilation was seen in the patients (r = 0.88, p less than 0.05), i.e., an optimal minute ventilation was shown. These relationships may be important when designing future clinical studies.« less

Face Masks and Cough Etiquette Reduce the Cough Aerosol Concentration of Pseudomonas aeruginosa in People with Cystic Fibrosis.

PubMed

Wood, Michelle E; Stockwell, Rebecca E; Johnson, Graham R; Ramsay, Kay A; Sherrard, Laura J; Jabbour, Nassib; Ballard, Emma; O'Rourke, Peter; Kidd, Timothy J; Wainwright, Claire E; Knibbs, Luke D; Sly, Peter D; Morawska, Lidia; Bell, Scott C

2018-02-01

People with cystic fibrosis (CF) generate Pseudomonas aeruginosa in droplet nuclei during coughing. The use of surgical masks has been recommended in healthcare settings to minimize pathogen transmission between patients with CF. To determine if face masks and cough etiquette reduce viable P. aeruginosa aerosolized during coughing. Twenty-five adults with CF and chronic P. aeruginosa infection were recruited. Participants performed six talking and coughing maneuvers, with or without face masks (surgical and N95) and hand covering the mouth when coughing (cough etiquette) in an aerosol-sampling device. An Andersen Cascade Impactor was used to sample the aerosol at 2 meters from each participant. Quantitative sputum and aerosol bacterial cultures were performed, and participants rated the mask comfort levels during the cough maneuvers. During uncovered coughing (reference maneuver), 19 of 25 (76%) participants produced aerosols containing P. aeruginosa, with a positive correlation found between sputum P. aeruginosa concentration (measured as cfu/ml) and aerosol P. aeruginosa colony-forming units. There was a reduction in aerosol P. aeruginosa load during coughing with a surgical mask, coughing with an N95 mask, and cough etiquette compared with uncovered coughing (P < 0.001). A similar reduction in total colony-forming units was observed for both masks during coughing; yet, participants rated the surgical masks as more comfortable (P = 0.013). Cough etiquette provided approximately half the reduction of viable aerosols of the mask interventions during voluntary coughing. Talking was a low viable aerosol-producing activity. Face masks reduce cough-generated P. aeruginosa aerosols, with the surgical mask providing enhanced comfort. Cough etiquette was less effective at reducing viable aerosols.

The effects of inhaled aztreonam on the cystic fibrosis lung microbiome.

PubMed

Heirali, Alya A; Workentine, Matthew L; Acosta, Nicole; Poonja, Ali; Storey, Douglas G; Somayaji, Ranjani; Rabin, Harvey R; Whelan, Fiona J; Surette, Michael G; Parkins, Michael D

2017-05-05

Aztreonam lysine for inhalation (AZLI) is an inhaled antibiotic used to treat chronic Pseudomonas aeruginosa infection in CF. AZLI improves lung function and quality of life, and reduces exacerbations-improvements attributed to its antipseudomonal activity. Given the extremely high aztreonam concentrations achieved in the lower airways by nebulization, we speculate this may extend its spectrum of activity to other organisms. As such, we sought to determine if AZLI affects the CF lung microbiome and whether community constituents can be used to predict treatment responsiveness. Patients were included if they had chronic P. aeruginosa infection and repeated sputum samples collected before and after AZLI. Sputum DNA was extracted, and the V3-hypervariable region of the 16S ribosomal RNA (rRNA) gene amplified and sequenced. Twenty-four patients naïve to AZLI contributed 162 samples. The cohort had a median age of 37.1 years, and a  median FEV 1 of 44% predicted. Fourteen patients were a priori defined as responders for achieving ≥3% FEV 1 improvement following initiation. No significant changes in alpha diversity were noted following AZLI. Furthermore, beta diversity demonstrated clustering with respect to patients, but had no association with AZLI use. However, we did observe a decline in the relative abundance of several individual operational taxonomic units (OTUs) following AZLI initiation suggesting that specific sub-populations of organisms may be impacted. Patients with higher abundance of Staphylococcus and anaerobic organisms including Prevotella and Fusobacterium were less likely to respond to therapy. Results from our study suggest potential alternate/additional mechanisms by which AZLI functions. Moreover, our study suggests that the CF microbiota may be used as a biomarker to predict patient responsiveness to therapy suggesting the microbiome may be harnessed for the personalization of therapies.

In vitro activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia lifestyles under conditions relevant to pulmonary infection in cystic fibrosis, and relationship with SmeDEF multidrug efflux pump expression.

PubMed

Pompilio, Arianna; Crocetta, Valentina; Verginelli, Fabio; Di Bonaventura, Giovanni

2016-07-01

The activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia cells and the role played by the multidrug efflux pump SmeDEF were evaluated under conditions relevant to the cystic fibrosis (CF) lung. MIC, MBC and MBEC of levofloxacin were assessed, against five CF strains, under 'standard' (CLSI-recommended) and 'CF-like' (pH 6.8, 5% CO2, in a synthetic CF sputum) conditions. Levofloxacin was tested against biofilms at concentrations (10, 50 and 100 μg mL(-1)) corresponding to achievable serum levels and sputum levels by aerosolisation. smeD expression was evaluated, under both conditions, in planktonic and biofilm cells by RT-PCR. The bactericidal effect of levofloxacin was decreased, in three out of five strains tested, under 'CF-like' conditions (MBC: 2-4 vs 8-16 μg mL(-1), under 'standard' and 'CF-like' conditions, respectively). Biofilm was intrinsically resistant to levofloxacin, regardless of conditions tested (MBECs ≥ 100 μg mL(-1) for all strains). Only under 'CF-like' conditions, smeD expression increased during planktonic-to-biofilm transition, and in biofilm cells compared to stationary planktonic cells. Our findings confirmed that S. maltophilia biofilm is intrinsically resistant to therapeutic concentrations of levofloxacin. Under conditions relevant to CF, smeD overexpression could contribute to levofloxacin resistance. Further studies are warranted to define the clinical relevance of our findings. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Use of Selective Fungal Culture Media Increases Rates of Detection of Fungi in the Respiratory Tract of Cystic Fibrosis Patients.

PubMed

Hong, Gina; Miller, Heather B; Allgood, Sarah; Lee, Richard; Lechtzin, Noah; Zhang, Sean X

2017-04-01

The prevalence of fungi in the respiratory tracts of cystic fibrosis (CF) patients has risen. However, fungal surveillance is not routinely performed in most clinical centers in the United States, which may lead to an underestimation of the true prevalence of the problem. We conducted a prospective study comparing the rates of detection for clinically important fungi (CIF), defined as Aspergillus , Scedosporium , and Trichosporon species and Exophiala dermatitidis , in CF sputa using standard bacterial and selective fungal culture media, including Sabouraud dextrose agar with gentamicin (SDA), inhibitory mold agar (IMA), and brain heart infusion (BHI) agar with chloramphenicol and gentamicin. We described the prevalence of these fungi in an adult CF population. A total of 487 CF respiratory samples were collected from 211 unique participants. CIF were detected in 184 (37.8%) samples. Only 26.1% of CIF-positive samples were detected in bacterial culture medium, whereas greater rates of detection for fungi were found in IMA (65.8%; P < 0.001), in SDA (at 30°C, 64.7%; P = 0.005), and in BHI agar (63.0%; P = 0.001). The prevalences of Aspergillus and Scedosporium species were 40.8% and 5.2%, respectively, which are greater than the nationally reported prevalence numbers of 20.4% and 1.9%. Selective fungal culture media and longer incubation periods yielded higher rates of detection for CIF in CF sputum samples compared with that detected in bacterial culture medium, resulting in an underdetection of fungi by bacterial culture alone. The prevalence of fungi in CF may be better estimated by using selective fungal culture media, and this may translate to important clinical decisions. Copyright © 2017 American Society for Microbiology.

Use of Selective Fungal Culture Media Increases Rates of Detection of Fungi in the Respiratory Tract of Cystic Fibrosis Patients

PubMed Central

Hong, Gina; Miller, Heather B.; Allgood, Sarah; Lee, Richard; Lechtzin, Noah

2017-01-01

ABSTRACT The prevalence of fungi in the respiratory tracts of cystic fibrosis (CF) patients has risen. However, fungal surveillance is not routinely performed in most clinical centers in the United States, which may lead to an underestimation of the true prevalence of the problem. We conducted a prospective study comparing the rates of detection for clinically important fungi (CIF), defined as Aspergillus, Scedosporium, and Trichosporon species and Exophiala dermatitidis, in CF sputa using standard bacterial and selective fungal culture media, including Sabouraud dextrose agar with gentamicin (SDA), inhibitory mold agar (IMA), and brain heart infusion (BHI) agar with chloramphenicol and gentamicin. We described the prevalence of these fungi in an adult CF population. A total of 487 CF respiratory samples were collected from 211 unique participants. CIF were detected in 184 (37.8%) samples. Only 26.1% of CIF-positive samples were detected in bacterial culture medium, whereas greater rates of detection for fungi were found in IMA (65.8%; P < 0.001), in SDA (at 30°C, 64.7%; P = 0.005), and in BHI agar (63.0%; P = 0.001). The prevalences of Aspergillus and Scedosporium species were 40.8% and 5.2%, respectively, which are greater than the nationally reported prevalence numbers of 20.4% and 1.9%. Selective fungal culture media and longer incubation periods yielded higher rates of detection for CIF in CF sputum samples compared with that detected in bacterial culture medium, resulting in an underdetection of fungi by bacterial culture alone. The prevalence of fungi in CF may be better estimated by using selective fungal culture media, and this may translate to important clinical decisions. PMID:28100601

Effects of Autogenic Drainage on Sputum Recovery and Pulmonary Function in People with Cystic Fibrosis: A Systematic Review.

PubMed

Morgan, Kimbly; Osterling, Kristin; Gilbert, Robert; Dechman, Gail

2015-01-01

To determine the effects of short- and long-term use of autogenic drainage (AD) on pulmonary function and sputum recovery in people with cystic fibrosis (CF). The authors conducted a systematic review of randomized and quasi-randomized clinical trials in which participants were people with CF who use AD as their sole airway clearance technique. Searches in 4 databases and secondary sources using 5 key terms yielded 735 articles, of which 58 contained the terms autogenic drainage and cystic fibrosis. Ultimately, 4 studies, 2 of which were long term, were included. All measured forced expiratory volume in 1 second (FEV1) and found no change. The long-term studies were underpowered to detect change in FEV1; however, the short-term studies found a clinically significant sputum yield (≥4 g). AD has been shown to produce clinically significant sputum yields in a limited number of investigations. The effect of AD on the function of the pulmonary system remains uncertain, and questions have emerged regarding the appropriateness of FEV1 as a valid measure of airway clearance from peripheral lung regions. Further consideration should be given to the use of FEV1 as a primary measure of the effect of AD.

Effects of Autogenic Drainage on Sputum Recovery and Pulmonary Function in People with Cystic Fibrosis: A Systematic Review

PubMed Central

Osterling, Kristin; Gilbert, Robert; Dechman, Gail

2015-01-01

ABSTRACT Purpose: To determine the effects of short- and long-term use of autogenic drainage (AD) on pulmonary function and sputum recovery in people with cystic fibrosis (CF). Methods: The authors conducted a systematic review of randomized and quasi-randomized clinical trials in which participants were people with CF who use AD as their sole airway clearance technique. Results: Searches in 4 databases and secondary sources using 5 key terms yielded 735 articles, of which 58 contained the terms autogenic drainage and cystic fibrosis. Ultimately, 4 studies, 2 of which were long term, were included. All measured forced expiratory volume in 1 second (FEV1) and found no change. The long-term studies were underpowered to detect change in FEV1; however, the short-term studies found a clinically significant sputum yield (≥4 g). Conclusion: AD has been shown to produce clinically significant sputum yields in a limited number of investigations. The effect of AD on the function of the pulmonary system remains uncertain, and questions have emerged regarding the appropriateness of FEV1 as a valid measure of airway clearance from peripheral lung regions. Further consideration should be given to the use of FEV1 as a primary measure of the effect of AD. PMID:27504031

Eradication failure of newly acquired Pseudomonas aeruginosa isolates in cystic fibrosis.

PubMed

Cohen-Cymberknoh, Malena; Gilead, Noa; Gartner, Silvia; Rovira, Sandra; Blau, Hannah; Mussaffi, Huda; Rivlin, Joseph; Gur, Michal; Shteinberg, Michal; Bentur, Lea; Livnat, Galit; Aviram, Micha; Picard, Elie; Tenenbaum, Ariel; Armoni, Shoshana; Breuer, Oded; Shoseyov, David; Kerem, Eitan

2016-11-01

Eradication of Pseudomonas aeruginosa (PA) is critical in cystic fibrosis (CF) patients. To determine eradication success rate of newly acquired PA and to identify characteristics associated with eradication failure. In an observational study, data from patients with newly acquired PA infection from 2007 to 2013 were collected. Clinical variables were compared in patients with and without successful eradication for ≥1year. Of 183 patients out of 740 (25%) from 7 CF Centers that had newly acquired PA, eradication succeeded in 72%. Patients with the highest risk of failure had multi-resistant PA, fewer sputum cultures taken, were older, and were diagnosed at a later age. The risk of eradication failure increased by 1.3% with each year of delayed CF diagnosis; successful eradication increased by 17% with each additional sputum culture taken. Delayed detection of PA infection leading to delayed treatment and growth of multi-resistant organisms is associated with eradication failure. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Data Mining of Lung Microbiota in Cystic Fibrosis Patients.

PubMed

Li, Jianguo; Hao, Chunyan; Ren, Lili; Xiao, Yan; Wang, Jianwei; Qin, Xuemei

2016-01-01

The major therapeutic strategy used to treat exacerbated cystic fibrosis (CF) is antibiotic treatment. As this approach easily generates antibiotic-resistant strains of opportunistic bacteria, optimized antibiotic therapies are required to effectively control chronic and recurrent bacterial infections in CF patients. A promising future for the proper use of antibiotics is the management of lung microbiota. However, the impact of antibiotic treatments on CF microbiota and vice versa is not fully understood. This study analyzed 718 sputum samples from 18 previous studies to identify differences between CF and uninfected lung microbiota and to evaluate the effects of antibiotic treatments on exacerbated CF microbiota. A reference-based OTU (operational taxonomic unit) picking method was used to combine analyses of data generated using different protocols and platforms. Findings show that CF microbiota had greater richness and lower diversity in the community structure than uninfected control (NIC) microbiota. Specifically, CF microbiota showed higher levels of opportunistic bacteria and dramatically lower levels of commensal bacteria. Antibiotic treatment affected exacerbated CF microbiota notably but only transiently during the treatment period. Limited decrease of the dominant opportunistic bacteria and a dramatic decrease of commensal bacteria were observed during the antibiotic treatment for CF exacerbation. Simultaneously, low abundance opportunistic bacteria were thriving after the antibiotic treatment. The inefficiency of the current antibiotic treatment against major opportunistic bacteria and the detrimental effects on commensal bacteria indicate that the current empiric antibiotic treatment on CF exacerbation should be reevaluated and optimized.

Data Mining of Lung Microbiota in Cystic Fibrosis Patients

PubMed Central

Xiao, Yan; Wang, Jianwei; Qin, Xuemei

2016-01-01

The major therapeutic strategy used to treat exacerbated cystic fibrosis (CF) is antibiotic treatment. As this approach easily generates antibiotic-resistant strains of opportunistic bacteria, optimized antibiotic therapies are required to effectively control chronic and recurrent bacterial infections in CF patients. A promising future for the proper use of antibiotics is the management of lung microbiota. However, the impact of antibiotic treatments on CF microbiota and vice versa is not fully understood. This study analyzed 718 sputum samples from 18 previous studies to identify differences between CF and uninfected lung microbiota and to evaluate the effects of antibiotic treatments on exacerbated CF microbiota. A reference-based OTU (operational taxonomic unit) picking method was used to combine analyses of data generated using different protocols and platforms. Findings show that CF microbiota had greater richness and lower diversity in the community structure than uninfected control (NIC) microbiota. Specifically, CF microbiota showed higher levels of opportunistic bacteria and dramatically lower levels of commensal bacteria. Antibiotic treatment affected exacerbated CF microbiota notably but only transiently during the treatment period. Limited decrease of the dominant opportunistic bacteria and a dramatic decrease of commensal bacteria were observed during the antibiotic treatment for CF exacerbation. Simultaneously, low abundance opportunistic bacteria were thriving after the antibiotic treatment. The inefficiency of the current antibiotic treatment against major opportunistic bacteria and the detrimental effects on commensal bacteria indicate that the current empiric antibiotic treatment on CF exacerbation should be reevaluated and optimized. PMID:27741283

Progression of pulmonary disease after disappearance of Pseudomonas in cystic fibrosis.

PubMed

Sharma, G D; Tosi, M F; Stern, R C; Davis, P B

1995-07-01

Once cystic fibrosis (CF) patients become chronically colonized, eradication of Pseudomonas aeruginosa (PA) is rare. We report five patients, each colonized for at least 6 yr, whose subsequent cultures did not reveal PA or any other gram-negative pathogen for at least 2 yr. Two patients harbored yeast, normal throat flora, and occasional colonies of Aspergillus fumigatus, but no PA, Haemophilus influenzae, or other gram-negative pathogens. In two patients, sputum cultures revealed Staphylococcus aureus. Sputum smear showed no gram-negative organisms in any patient. Antipseudomonal antibody titers in all patients decreased during the noncolonized period. However, titers increased in four other CF patients who had continued PA colonization. Despite disappearance of PA, four of five patients had clinical progression of lung disease with deterioration of FEV1. We conclude that (1) PA, even after it has chronically colonized the airways of CF patients, can occasionally disappear; and (2) lung disease can progress despite the disappearance of PA. Thus, the elimination of a milieu favorable to PA, such as might be anticipated with therapy directed at the basic defect, may not be sufficient to halt the pulmonary disease progression in CF.

Phenotypic and Genetic Characterization of Carbapenemase and ESBLs Producing Gram-negative Bacteria (GNB) Isolated from Patients with Cystic Fibrosis (CF) in Tehran Hospitals

PubMed Central

Vali, Parisa; Shahcheraghi, Fereshteh; Seyfipour, Maryam; Zamani, Maryam Alsadat; Allahyar, Mohammad Reza; Feizabadi, Mohammad Mehdi

2014-01-01

Background: Cystic Fibrosis (CF) is an autosomal recessive genetic disorder in white populations caused by mutation in a gene that encodes Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Since frequent respiratory tract infections are the major problem in patients with CF, obligation to identify the causative bacteria and determining their antibiotic resistance pattern is crucial. The purpose of this project was to detect Gram-negative bacteria (GNB) isolated from sputa of CF patients and to determine their antibiotic resistance pattern. Materials and Methods: The sputum of 52 CF patients, treated as inpatients at hospitals in Tehran, was obtained between November 2011 and June 2012. Samples cultured in selective and non-selective media and GNB recognized by biochemical tests. Antimicrobial susceptibility testing to cephalosporins, aminoglycosides and carbapenems was performed by disk diffusion method and MICs of them were measured. For phenotypic detection of carbapenemase and ESBLs production, the Modified Hodge test, double disk synergy test and the combined disk methods were performed. Subsequently, the genes encoding the extended spectrum beta-lactamases (blaPER, blaCTX-M) and carbapenemases (blaIMP-1, blaGES, blaKPC, blaNDM, blaVIM-1, blaVIM-2, blaSPM, blaSIM) in Gram negative bacteria were targeted among the resistant isolates by using PCR. PFGE was used to determine any genetic relationship among the Pseudomonas aeruginosa isolated from these patients. Results: Fifty five GNB were isolated from 52 sputum samples including Pseudomonas aeruginosa, Klebsiella ozaenae, Alcaligenes xylosoxidans, Achromobacter denitrificans, Klebsiella pneumonia and Stenotrophomonas maltophilia. The rates of resistance to different antibiotic were as follows: cefixime (%80), ceftriaxone (%43), ceftazidime (%45) and meropenem (%7). The prevalence of genes encoding the ESBLs and Carbapenemases among the the phenotypically positive strains were as follows: blaCTX-M (19), blaIMP-1 (2), blaVIM-1 (2) and blaVIM-2 (3) genes respectively. No other genes were detected. PFGE analysis revealed 8 genotypes. Six isolates had mutually 3 similar patterns. Conclusion: This study showed the existence of important ESBLs and carbapenemases genes among the GNB isolated from patients with CF. Continuous surveillance of ESBLs and Carbapenemases, also identification of their types, in bacteria isolated from these patients have an important clinical impact, since, it can often provide valuable information for effective infection control measures and for the choice of appropriate antimicrobial therapy. PMID:24596716

Individual Patterns of Complexity in Cystic Fibrosis Lung Microbiota, Including Predator Bacteria, over a 1-Year Period.

PubMed

de Dios Caballero, Juan; Vida, Rafael; Cobo, Marta; Máiz, Luis; Suárez, Lucrecia; Galeano, Javier; Baquero, Fernando; Cantón, Rafael; Del Campo, Rosa

2017-09-26

Cystic fibrosis (CF) lung microbiota composition has recently been redefined by the application of next-generation sequencing (NGS) tools, identifying, among others, previously undescribed anaerobic and uncultivable bacteria. In the present study, we monitored the fluctuations of this ecosystem in 15 CF patients during a 1-year follow-up period, describing for the first time, as far as we know, the presence of predator bacteria in the CF lung microbiome. In addition, a new computational model was developed to ascertain the hypothetical ecological repercussions of a prey-predator interaction in CF lung microbial communities. Fifteen adult CF patients, stratified according to their pulmonary function into mild ( n = 5), moderate ( n = 9), and severe ( n = 1) disease, were recruited at the CF unit of the Ramón y Cajal University Hospital (Madrid, Spain). Each patient contributed three or four induced sputum samples during a 1-year follow-up period. Lung microbiota composition was determined by both cultivation and NGS techniques and was compared with the patients' clinical variables. Results revealed a particular microbiota composition for each patient that was maintained during the study period, although some fluctuations were detected without any clinical correlation. For the first time, Bdellovibrio and Vampirovibrio predator bacteria were shown in CF lung microbiota and reduced-genome bacterial parasites of the phylum Parcubacteria were also consistently detected. The newly designed computational model allows us to hypothesize that inoculation of predators into the pulmonary microbiome might contribute to the control of chronic colonization by CF pathogens in early colonization stages. IMPORTANCE The application of NGS to sequential samples of CF patients demonstrated the complexity of the organisms present in the lung (156 species) and the constancy of basic individual colonization patterns, although some differences between samples from the same patient were observed, probably related to sampling bias. Bdellovibrio and Vampirovibrio predator bacteria were found for the first time by NGS as part of the CF lung microbiota, although their ecological significance needs to be clarified. The newly designed computational model allows us to hypothesize that inoculation of predators into the lung microbiome can eradicate CF pathogens in early stages of the process. Our data strongly suggest that lower respiratory microbiome fluctuations are not necessarily related to the patient's clinical status. Copyright © 2017 de Dios Caballero et al.

Duplication in CHIT1 gene and the risk for Aspergillus lung disease in CF patients.

PubMed

Livnat, Galit; Bar-Yoseph, Ronen; Mory, Adi; Dagan, Efrat; Elias, Nael; Gershoni, Ruth; Bentur, Lea

2014-01-01

Aspergillus often persists in the respiratory tract of patients with Cystic Fibrosis (CF) and may cause allergic broncho-pulmonary aspergillosis (ABPA). Chitinases are enzymes that digest the chitin polymer. Plants use chitinase as a defense mechanism against fungi. Chitotriosidase (CHIT1) is the major chitinase in human airways. Variation in the coding region with 24-bp duplication allele results in reduced CHIT1 activity. Recently, CHIT1 duplication heterozygocity was found in 6/6 patients with severe asthma and fungal sensitization (SAFS). Our aim was to evaluate the link between CHIT1 duplication in CF patients and the predisposition to Allergic broncho-pulmonary mycosis (ABPM) or persistent Aspergillus positive sputum (APS). CHIT1 duplication was assessed in three CF groups. Group 1: patients who had neither ABPM nor APS in the past (control group). Group 2: patients with persistent APS (≥2/year), without ABPA. Group 3: patients with current or past ABPM. Forty patients with CF were included in the analysis, CHIT1 duplication heterozygocity was found in 3/6 (50%) of the patients in the ABPM group, 3/12 (25%) in the APS group, and 7/22 (31.8%) in the control group (P > 0.05). Eleven patients carried W1282X mutation, 90.9% were negative for CHIT1 duplication, five of them were homozygous for W1282X; none of them had CHIT1 duplication or ABPM. CHIT1 duplication is not found in all CF patients with ABPM in contrast to patients with SAFS. These results suggest that CHIT1 duplication cannot be the sole explanation for Aspergillus positive sputum in CF patients. © 2013 Wiley Periodicals, Inc.

Treatment of Aspergillus fumigatus in patients with cystic fibrosis: a randomized, placebo-controlled pilot study.

PubMed

Aaron, Shawn D; Vandemheen, Katherine L; Freitag, Andreas; Pedder, Linda; Cameron, William; Lavoie, Annick; Paterson, Nigel; Wilcox, Pearce; Rabin, Harvey; Tullis, Elizabeth; Morrison, Nancy; Ratjen, Felix

2012-01-01

Many patients with cystic fibrosis develop persistent airway infection/colonization with Aspergillus fumigatus, however the impact of A. fumigatus on clinical outcomes remains unclear. The objective of this study was to determine whether treatment directed against Aspergillus fumigatus improves pulmonary function and clinical outcomes in patients with cystic fibrosis (CF). We performed a double-blind randomized placebo-controlled pilot clinical trial involving 35 patients with CF whose sputum cultures were chronically positive for A. fumigatus. Participants were centrally randomized to receive either oral itraconazole 5 mg/kg/d (N = 18) or placebo (N = 17) for 24 weeks. The primary outcome was the proportion of patients who experienced a respiratory exacerbation requiring intravenous antibiotics over the 24 week treatment period. Secondary outcomes included changes in FEV(1) and quality of life. Over the 24 week treatment period, 4 of 18 (22%) patients randomized to itraconazole experienced a respiratory exacerbation requiring intravenous antibiotics, compared to 5 of 16 (31%) placebo treated patients, P = 0.70. FEV(1) declined by 4.62% over 24 weeks in the patients randomized to itraconazole, compared to a 0.32% improvement in the placebo group (between group difference = -4.94%, 95% CI: -15.33 to 5.45, P = 0.34). Quality of life did not differ between the 2 treatment groups throughout the study. Therapeutic itraconazole blood levels were not achieved in 43% of patients randomized to itraconazole. We did not identify clinical benefit from itraconazole treatment for CF patients whose sputum was chronically colonized with A. fumigatus. Limitations of this pilot study were its small sample size, and failure to achieve therapeutic levels of itraconazole in many patients. ClinicalTrials.gov NCT00528190.

Individual Patterns of Complexity in Cystic Fibrosis Lung Microbiota, Including Predator Bacteria, over a 1-Year Period

PubMed Central

de Dios Caballero, Juan; Vida, Rafael; Cobo, Marta; Máiz, Luis; Suárez, Lucrecia; Galeano, Javier; Baquero, Fernando; Cantón, Rafael

2017-01-01

ABSTRACT Cystic fibrosis (CF) lung microbiota composition has recently been redefined by the application of next-generation sequencing (NGS) tools, identifying, among others, previously undescribed anaerobic and uncultivable bacteria. In the present study, we monitored the fluctuations of this ecosystem in 15 CF patients during a 1-year follow-up period, describing for the first time, as far as we know, the presence of predator bacteria in the CF lung microbiome. In addition, a new computational model was developed to ascertain the hypothetical ecological repercussions of a prey-predator interaction in CF lung microbial communities. Fifteen adult CF patients, stratified according to their pulmonary function into mild (n = 5), moderate (n = 9), and severe (n = 1) disease, were recruited at the CF unit of the Ramón y Cajal University Hospital (Madrid, Spain). Each patient contributed three or four induced sputum samples during a 1-year follow-up period. Lung microbiota composition was determined by both cultivation and NGS techniques and was compared with the patients’ clinical variables. Results revealed a particular microbiota composition for each patient that was maintained during the study period, although some fluctuations were detected without any clinical correlation. For the first time, Bdellovibrio and Vampirovibrio predator bacteria were shown in CF lung microbiota and reduced-genome bacterial parasites of the phylum Parcubacteria were also consistently detected. The newly designed computational model allows us to hypothesize that inoculation of predators into the pulmonary microbiome might contribute to the control of chronic colonization by CF pathogens in early colonization stages. PMID:28951476

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

PubMed Central

Rogers, Geraint B; Daniels, Thomas WV; Tuck, Andrew; Carroll, Mary P; Connett, Gary J; David, Gondi JP; Bruce, Kenneth D

2009-01-01

Background Drawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment. Methods Six different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches. Results A mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures. Conclusion Due to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant. PMID:19368727

Insufficient quality of sputum submitted for tuberculosis diagnosis and associated factors, in Klaten district, Indonesia

PubMed Central

Sakundarno, Mateus; Nurjazuli, Nurjazuli; Jati, Sutopo Patria; Sariningdyah, Retna; Purwadi, Sumarsono; Alisjahbana, Bachti; van der Werf, Marieke J

2009-01-01

Background Sputum smear microscopy is the standard diagnostic method for detection of smear positive pulmonary tuberculosis (TB). Insufficient quality of sputum might result in missing cases. In this study we aimed at assessing the quality of sputum in a district in Central Java and determining patient and health worker factors associated with submission of three good quality sputum samples. Methods In 16 health centers information was collected on the quality of sputum submitted by TB suspects, i.e. volume, color, and viscosity. TB suspects were interviewed to assess their knowledge of TB, motivation to provide sputum and whether they were informed why and how to produce a sputum sample. Health workers were interviewed to assess what information they provided to TB suspects about the reason for sputum examination, methods to produce sputum and characteristics of a good quality sputum sample. All health worker and patient factors were evaluated for association with sputum quality. Results Of 387 TB suspects, 294 (76.0%) could be traced and interviewed, and of 272 (70.3%) information about sputum quality was available. Of those 203 (74.6%) submitted three samples, 90 (33.1%) provided at least one good sample, and 37 (13.6%) provided three good quality sputum samples. Of the 272 TB suspects, 168 (61.8%) mentioned that information on the reason for sputum examination was provided, 66 (24.3%) remembered that they were informed about how to produce sputum and 40 (14.7%) recalled being informed about the characteristics of good quality sputum. Paramedics reported to provide often/always information on the importance of sputum examination, and when to produce sputum. Information on how to produce sputum and characteristics of a good sputum sample was less often provided. None of the studied patient characteristics or health worker factors was associated with providing good quality sputum. Conclusion A considerable number of TB suspects did not provide three sputum samples and a large number of sputum samples were of insufficient quality. Training of health workers in providing health education to the TB suspect about the reason for sputum examination and how to produce a good quality sputum sample should be a priority of the TB program. PMID:19426477

Audit of the practice of sputum smear examination for patients with suspected pulmonary tuberculosis in Fiji.

PubMed

Gounder, Shakti; Tayler-Smith, Katherine; Khogali, Mohammed; Raikabula, Maopa; Harries, Anthony D

2013-07-01

In Fiji, patients with suspected pulmonary tuberculosis (PTB) currently submit three sputum specimens for smear microscopy for acid-fast bacilli, but there is little information about how well this practice is carried out. A cross-sectional retrospective review was carried out in all four TB diagnostic laboratories in Fiji to determine among new patients presenting with suspected PTB in 2011: the quality of submitted sputum; the number of sputum samples submitted; the relationship between quality and number of submitted samples to smear-positivity; and positive yield from first, second and third samples. Of 1940 patients with suspected PTB, 3522 sputum samples were submitted: 997 (51.4%) patients submitted one sample, 304 (15.7%) patients submitted two samples and 639 (32.9%) submitted three samples. Sputum quality was recorded in 2528 (71.8%) of samples, of which 1046 (41.4%) were of poor quality. Poor quality sputum was more frequent in females, inpatients and children (0-14 years). Good quality sputum and a higher number of submitted samples positively correlated with smear-positivity for acid-fast bacilli. There were 122 (6.3%) patients with suspected PTB who were sputum smear positive. Of those, 89 had submitted three sputum samples: 79 (89%) were diagnosed based on the first sputum sample, 6 (7%) on the second sample and 4 (4%) on the third sample. This study shows that there are deficiencies in the practice of sputum smear examination in Fiji with respect to sputum quality and recommended number of submitted samples, although the results support the continued use of three sputum samples for TB diagnosis. Ways to improve sputum quality and adherence to recommended guidelines are needed.

In Vitro Analysis of Pseudomonas aeruginosa Virulence Using Conditions That Mimic the Environment at Specific Infection Sites.

PubMed

Colmer-Hamood, J A; Dzvova, N; Kruczek, C; Hamood, A N

2016-01-01

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes chronic lung infection in patients with cystic fibrosis (CF) and acute systemic infections in severely burned patients and immunocompromised patients including cancer patients undergoing chemotherapy and HIV infected individuals. In response to the environmental conditions at specific infection sites, P. aeruginosa expresses certain sets of cell-associated and extracellular virulence factors that produce tissue damage. Analyzing the mechanisms that govern the production of these virulence factors in vitro requires media that closely mimic the environmental conditions within the infection sites. In this chapter, we review studies based on media that closely resemble three in vivo conditions, the thick mucus accumulated within the lung alveoli of CF patients, the serum-rich wound bed and the bloodstream. Media resembling the CF alveolar mucus include standard laboratory media supplemented with sputum obtained from CF patients as well as prepared synthetic mucus media formulated to contain the individual components of CF sputum. Media supplemented with serum or individual serum components have served as surrogates for the soluble host components of wound infections, while whole blood has been used to investigate the adaptation of pathogens to the bloodstream. Studies using these media have provided valuable information regarding P. aeruginosa gene expression in different host environments as varying sets of genes were differentially regulated during growth in each medium. The unique effects observed indicate the essential role of these in vitro media that closely mimic the in vivo conditions in providing accurate information regarding the pathogenesis of P. aeruginosa infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Relative contribution of Prevotella intermedia and Pseudomonas aeruginosa to lung pathology in airways of patients with cystic fibrosis.

PubMed

Ulrich, Martina; Beer, Isabelle; Braitmaier, Peter; Dierkes, Michaela; Kummer, Florian; Krismer, Bernhard; Schumacher, Ulrike; Gräpler-Mainka, Ute; Riethmüller, Joachim; Jensen, Peter Ø; Bjarnsholt, Thomas; Høiby, Niels; Bellon, Gabriel; Döring, Gerd

2010-11-01

Patients with cystic fibrosis (CF) with Pseudomonas aeruginosa lung infections produce endobronchial mucus plugs allowing growth of obligate anaerobes including Prevotella spp. Whether obligate anaerobes contribute to the pathophysiology of CF lung disease is unknown. The virulence of Prevotella intermedia and Ps aeruginosa was investigated in vitro and in mice, antibodies against P intermedia in CF sera were assessed and a culture-independent detection method for P intermedia/P nigrescens in CF sputum was tested. P intermedia reached cell numbers of >10(5)->10(7) colony-forming units (CFU)/ml sputum. The majority of patients with CF (16/17; 94.1%) produced antibodies against two immunoreactive antigens of P intermedia. Culture supernatant fluids, collected from 10(9) P intermedia cells, were more cytotoxic to respiratory epithelial cells in vitro and inflammatory in mouse lungs than respective fluids from anaerobically grown Ps aeruginosa, while fluids from aerobically grown Ps aeruginosa had the highest cytotoxicity and inflammation. Both pathological effects were largely reduced when culture supernatant fluids from 10(7) cells of either species were used. P intermedia cells (∼10(6)CFU/lung) did not induce mortality in the agar beads lung infection mouse model, while Ps aeruginosa cells caused death in 30% of mice due to rapid multiplication. A P intermedia/P nigrescens-specific PNA probe was significantly more sensitive than culture-dependent diagnostic assays to detect these strict anaerobes. Ps aeruginosa and P intermedia become significantly virulent in vitro and in vivo when cell numbers exceed 10(8) CFU/lung.

Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov., sp. nov.

PubMed

Coenye, Tom; Goris, Johan; Spilker, Theodore; Vandamme, Peter; LiPuma, John J

2002-06-01

Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include Acinetobacter sp., Bordetella hinzii, Burkholderia fungorum, Comamonas testosteroni, Chryseobacterium sp., Herbaspirillum sp., Moraxella osloensis, Pandoraea genomospecies 4, Ralstonia gilardii, Ralstonia mannitolilytica, Rhizobium radiobacter, and Xanthomonas sp. In addition, one isolate most likely represents a novel Ralstonia species, whereas nine isolates belong to novel taxa within the alpha-PROTEOBACTERIA: Eight of these latter isolates are classified into the novel genus Inquilinus gen. nov. as Inquilinus limosus gen. nov., sp. nov., or as Inquilinus sp. The remaining 17 isolates are characterized as members of the family ENTEROBACTERIACEAE: The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.

Sphingobacterium respiratory tract infection in patients with cystic fibrosis

PubMed Central

2009-01-01

Background Bacteria that belong to the genus Sphingobacterium are Gram-negative, non-fermentative bacilli, ubiquitous in nature and rarely involved in human infections. The aims of this study were to evaluate the epidemiology of infection by Sphingobacterium in a cohort of patients affected by Cystic Fibrosis (CF), the antibiotic susceptibility and the DNA fingerprinting of the isolated strains and to analyze some clinical outcomes of the infected patients. Findings Between January 2006 and June 2008, patients (n = 332) attending the Regional CF Unit in Naples, Italy, were enrolled. Sputum samples were processed for microscopic, cultural, phenotypic identification and antibiotic susceptibility testing. DNA fingerprinting was performed by pulsed-field gel electrophoresis (PFGE). A total of 21 strains of Sphingobacterium were isolated from 7 patients (13 of S. spiritovorum, 8 of S. multivorum). S. multivorum isolates were more resistant than those of S. spiritovorum. PFGE profiles were in general heterogeneous, which suggested independent circulation. Conclusions This is the first Italian report about respiratory tract infections by Sphingobacterium in CF patients. In our cohort, these infections were not associated with a deterioration of pulmonary function during the follow-up period. Although the exact role of this microorganism in CF lung disease is unknown and the number of infected patients was small, this study could represent an important starting-point for understanding the epidemiology and the possible pathogenic role of Sphingobacterium in CF patients. PMID:20030840

The L441P mutation of cystic fibrosis transmembrane conductance regulator and its molecular pathogenic mechanisms in a Korean patient with cystic fibrosis.

PubMed

Gee, Heon Yung; Kim, Chang Keun; Kim, So Won; Lee, Ji Hyun; Kim, Jeong-Ho; Kim, Kyung Hwan; Lee, Min Goo

2010-01-01

Cystic fibrosis (CF) is an autosomal recessive disorder usually found in populations of white Caucasian descent. CF is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. A 5-yr-old Korean girl was admitted complaining of coughing and greenish sputum. Chest radiographs and computed tomographic (CT) scan revealed diffuse bronchiectasis in both lungs. The patient had chronic diarrhea and poor weight gain, and the abdominal pancreaticobiliary CT scan revealed atrophy of the pancreas. Finally, CF was confirmed by the repeated analysis of the quantitative pilocarpine iontophoresis test. The chloride concentration of sweat samples taken from both forearms of the pateint was an average of 88.7 mM/L (normal value <40 mM/L). After a comprehensive search for mutations in the CFTR gene, the patient was found to carry the non-synonymous L441P mutation in one allele. Molecular physiologic analysis of the L441P mutation of CFTR revealed that the L441P mutation completely abolished the CFTR Cl(-) channel activity by disrupting proper protein folding and membrane trafficking of CFTR protein. These results confirmed the pathogenicity of the L441P mutation of CFTR circulating in the Korean population. The possibility of CF should be suspected in patients with chronic bronchiectasis, although the frequency of CF is relatively rare in East Asia.

Inhaled dry powder mannitol in children with cystic fibrosis: A randomised efficacy and safety trial.

PubMed

De Boeck, K; Haarman, E; Hull, J; Lands, L C; Moeller, A; Munck, A; Riethmüller, J; Tiddens, H; Volpi, S; Leadbetter, J; Charlton, B; Malfroot, A

2017-05-01

Inhaled mannitol has beneficial effects on lung function, mucociliary clearance, quality of life and sputum properties. This trial examined the efficacy of inhaled mannitol in children with cystic fibrosis (CF). The efficacy of inhaled mannitol in children with CF aged 6-17years was assessed in a phase 2, randomised, placebo-controlled crossover study. Subjects were randomly assigned to mannitol 400mg every 12h or matching placebo for 8weeks, followed by an 8week washout and an 8week period with the alternate treatment. The primary endpoint was the absolute change from baseline in ppFEV1 (percent predicted FEV1). A total of 92 subjects were studied, with a mean age of 12years and mean baseline ppFEV1 of 72.2%. During mannitol treatment ppFEV1 was 3.42% (p=0.004) higher compared to placebo or a 4.97% (p=0.005) relative difference; relative change from baseline FEF25-75 was 10.52% (p=0.013). During mannitol treatment, acute post-treatment sputum weight was higher (p=0.012). In pre-specified subgroups (rhDNase use, age, and disease severity), the treatment differences consistently favoured mannitol. The most common AEs were cough and pulmonary exacerbations. Pulmonary exacerbation AEs were approximately 30% lower in the mannitol group. In children with CF, inhaled mannitol was associated with significant improvements in lung function and sputum weight, irrespective of rhDNase use, age or disease severity. Inhaled mannitol was well tolerated and was associated with a reduced incidence of pulmonary exacerbation AEs. (Clinical Trials.Gov: NCT 01883531). Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Unusual Aspergillus species in patients with cystic fibrosis.

PubMed

Symoens, Françoise; Haase, Gerhard; Pihet, Marc; Carrere, Jacqueline; Beguin, Hugues; Degand, Nicolas; Mely, Laurent; Bouchara, Jean-Philippe

2010-11-01

Poorly sporulating Aspergillus isolates from patients with cystic fibrosis (CF) are generally identified in routine procedures as Aspergillus spp. In this study, we identified and characterized 11 isolates belonging to two unusual Aspergillus species of the section Fumigati (A. lentulus and Neosartorya pseudofischeri) recovered from four different patients. Aspergillus lentulus was found occasionally during a 10-year follow-up study of one CF patient colonized by A. fumigatus. Neosartorya pseudofischeri was isolated from three patients followed in different European hospitals. This species was recovered from two sputum samples of one patient, and from four successive samples of the two other patients, suggesting that it may be responsible for chronic colonization. Both species were isolated together with A. fumigatus. Isolates from both species did not grow at 50°C, and DNA sequence analysis, together with further morphological observations permitted identification at the species level. Growth at different temperatures and antifungal susceptibility were also investigated. All the isolates of N. pseudofischeri exhibited a very low susceptibility to voriconazole (VRZ) whereas a very low susceptibility to VRZ and amphotericin B was seen with the A. lentulus isolates.

Effects of long-term azithromycin therapy on airway oxidative stress markers in non-cystic fibrosis bronchiectasis.

PubMed

Diego, Afredo De; Milara, Javier; Martinez-Moragón, Eva; Palop, Marta; León, Montse; Cortijo, Julio

2013-10-01

To explore the effect of long-term therapy with azithromycin in regards to airway oxidative stress markers in exhaled breath condensate (EBC) of adult patients with stable non-cystic fibrosis (CF) bronchiectasis. Open-label prospective study of 30 patients randomized to azithromycin 250 mg three times per week during 3 months (16 patients) or control (14 patients). Primary outcome were changes in nitric oxide, 8-isoprostane, pH, nitrites and nitrates in EBC. Secondary outcomes were changes in exacerbation rates, dyspnoea (Borg scale), sputum volume (cc), sputum colour (15-point scale), bacterial infection, health-related quality of life (St George's Respiratory Questionnaire), lung function and radiological extension. Azithromycin produced a significant decrease in sputum volume (8.9 (1.8) mL vs 2.1 (3.4) mL) and number of exacerbations (0.1 (0.6) vs 1.2 (0.9)). Dyspnoea (0.4 (0.1) vs 0.1 (0.2)) and health-related quality of life also improved after therapy. However, oxidative stress markers in EBC, systemic inflammatory markers as well as functional respiratory tests did not differ from the control group after therapy. A post-hoc analysis comparing patients infected or not with Pseudomonas aeruginosa revealed that these effects were more pronounced in infected patients. In this subgroup, treatment was followed by a significant reduction in sputum volume, number of exacerbations, dyspnoea and St George's Respiratory Questionnaire total score. Of all airway oxidative stress markers, only nitrates in EBC were reduced after therapy. Long-term azythromicin treatment has some clinical benefits in patients with non-CF stable bronchiectasis, but it does not affect airway oxidative stress markers. © 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.

The SENSOR Study: Protocol for a Mixed-Methods Study of Self-Management Checks to Predict Exacerbations of Pseudomonas Aeruginosa in Patients with Long-Term Respiratory Conditions

PubMed Central

Roberts, Claire; Gunatilake, Samal; Storrar, Will; Elliott, Scott; Glaysher, Sharon; Green, Ben; Rule, Steven; Fogg, Carole; Dewey, Ann; Auton, Kevin A; Chauhan, Anoop J

2017-01-01

Background There are an estimated three million people in the United Kingdom with chronic obstructive pulmonary disease (COPD), and the incidence of bronchiectasis is estimated at around 0.1% but is more common in COPD and severe asthma. Both COPD and bronchiectasis are characterized by exacerbations in which bacteria play a central role. Pseudomonas aeruginosa is isolated from sputum samples from 4% to 15% of adults with COPD and is more likely to be isolated from patients with severe disease. Earlier detection of exacerbations may improve morbidity and mortality by expediting treatment. Aseptika Ltd has developed a system for patients to self-monitor important physiological measurements including levels of physical activity, peak flow, forced expiratory volume (FEV1), and biomarkers for P aeruginosa in sputum. Objective We aim to test this system in 20 participants with P aeruginosa colonization and 10 controls with Haemophilus influenzae. Methods We plan to recruit 30 adult participants with COPD or non-CF bronchiectasis who have cultured P aeruginosa or H influenzae during an exacerbation in the last 6 months. They must produce sputum on most days and should have been stable for 4 weeks prior to entry. Daily data collected will include symptoms, health care usage, medication, weight, FEV1, physical activity level, blood pressure, oxygen saturation, and temperature. Sputum and urine samples will be provided daily. These data will be analyzed to assess predictive value in detecting upcoming exacerbations. Qualitative data will be gathered through self-administered questionnaires and semistructured interviews to gather information on participant coping and their use of the technology involved. Results Recruitment has been completed and results from the study should be available at the end of 2017. Conclusions The SENSOR study aims to test a home-monitoring system in people with chronic airway infection and is currently underway. PMID:28526665

Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears.

PubMed

Lavania, Surabhi; Anthwal, Divya; Bhalla, Manpreet; Singh, Nagendra; Haldar, Sagarika; Tyagi, Jaya Sivaswami

2017-01-01

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing.

Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears

PubMed Central

Lavania, Surabhi; Anthwal, Divya; Bhalla, Manpreet; Singh, Nagendra; Haldar, Sagarika; Tyagi, Jaya Sivaswami

2017-01-01

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing. PMID:29216262

Evidence and Role for Bacterial Mucin Degradation in Cystic Fibrosis Airway Disease

PubMed Central

Flynn, Jeffrey M.; Niccum, David; Dunitz, Jordan M.

2016-01-01

Chronic lung infections in cystic fibrosis (CF) patients are composed of complex microbial communities that incite persistent inflammation and airway damage. Despite the high density of bacteria that colonize the lower airways, nutrient sources that sustain bacterial growth in vivo, and how those nutrients are derived, are not well characterized. In this study, we examined the possibility that mucins serve as an important carbon reservoir for the CF lung microbiota. While Pseudomonas aeruginosa was unable to efficiently utilize mucins in isolation, we found that anaerobic, mucin-fermenting bacteria could stimulate the robust growth of CF pathogens when provided intact mucins as a sole carbon source. 16S rRNA sequencing and enrichment culturing of sputum also identified that mucin-degrading anaerobes are ubiquitous in the airways of CF patients. The collective fermentative metabolism of these mucin-degrading communities in vitro generated amino acids and short chain fatty acids (propionate and acetate) during growth on mucin, and the same metabolites were also found in abundance within expectorated sputum. The significance of these findings was supported by in vivo P. aeruginosa gene expression, which revealed a heightened expression of genes required for the catabolism of propionate. Given that propionate is exclusively derived from bacterial fermentation, these data provide evidence for an important role of mucin fermenting bacteria in the carbon flux of the lower airways. More specifically, microorganisms typically defined as commensals may contribute to airway disease by degrading mucins, in turn providing nutrients for pathogens otherwise unable to efficiently obtain carbon in the lung. PMID:27548479

Photoactivatable fluorescent probes reveal heterogeneous nanoparticle permeation through biological gels at multiple scales

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schuster, Benjamin S.; Allan, Daniel B.; Kays, Joshua C.

Diffusion through biological gels is crucial for effective drug delivery using nanoparticles. Here, we demonstrate a new method to measure diffusivity over a large range of length scales – from tens of nanometers to tens of micrometers – using photoactivatable fluorescent nanoparticle probes. We have applied this method to investigate the length-scale dependent mobility of nanoparticles in fibrin gels and in sputum from patients with cystic fibrosis (CF). Nanoparticles composed of poly(lactic-co-glycolic acid), with polyethylene glycol coatings to resist bioadhesion, were internally labeled with caged rhodamine to make the particles photoactivatable. We activated particles within a region of sample usingmore » brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles in that region to become fluorescent. We imaged the subsequent spatiotemporal evolution in fluorescence intensity and observed the collective particle diffusion over tens of minutes and tens of micrometers. We also performed complementary multiple particle tracking experiments on the same particles, extending significantly the range over which particle motion and its heterogeneity can be observed. In fibrin gels, both methods showed an immobile fraction of particles and a mobile fraction that diffused over all measured length scales. In the CF sputum, particle diffusion was spatially heterogeneous and locally anisotropic but nevertheless typically led to unbounded transport extending tens of micrometers within tens of minutes. Lastly, these findings provide insight into the mesoscale architecture of these gels and its role in setting their permeability on physiologically relevant length scales, pointing toward strategies for improving nanoparticle drug delivery.« less

Photoactivatable fluorescent probes reveal heterogeneous nanoparticle permeation through biological gels at multiple scales

DOE PAGES

Schuster, Benjamin S.; Allan, Daniel B.; Kays, Joshua C.; ...

2017-05-31

Diffusion through biological gels is crucial for effective drug delivery using nanoparticles. Here, we demonstrate a new method to measure diffusivity over a large range of length scales – from tens of nanometers to tens of micrometers – using photoactivatable fluorescent nanoparticle probes. We have applied this method to investigate the length-scale dependent mobility of nanoparticles in fibrin gels and in sputum from patients with cystic fibrosis (CF). Nanoparticles composed of poly(lactic-co-glycolic acid), with polyethylene glycol coatings to resist bioadhesion, were internally labeled with caged rhodamine to make the particles photoactivatable. We activated particles within a region of sample usingmore » brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles in that region to become fluorescent. We imaged the subsequent spatiotemporal evolution in fluorescence intensity and observed the collective particle diffusion over tens of minutes and tens of micrometers. We also performed complementary multiple particle tracking experiments on the same particles, extending significantly the range over which particle motion and its heterogeneity can be observed. In fibrin gels, both methods showed an immobile fraction of particles and a mobile fraction that diffused over all measured length scales. In the CF sputum, particle diffusion was spatially heterogeneous and locally anisotropic but nevertheless typically led to unbounded transport extending tens of micrometers within tens of minutes. Lastly, these findings provide insight into the mesoscale architecture of these gels and its role in setting their permeability on physiologically relevant length scales, pointing toward strategies for improving nanoparticle drug delivery.« less

Exopolysaccharides produced by clinical strains belonging to the Burkholderia cepacia complex.

PubMed

Herasimenka, Yury; Cescutti, Paola; Impallomeni, Giuseppe; Campana, Silvia; Taccetti, Giovanni; Ravenni, Novella; Zanetti, Flavio; Rizzo, Roberto

2007-04-01

In the frame of a research line dedicated to better clarify the role of exopolysaccharides (EPS) in bacterial virulence, EPS produced by species of the Burkholderia cepacia complex (Bcc), namely Burkholderia multivorans, Burkholderia cenocepacia, and a Bcc member of undetermined genomovar, all isolated at the Cystic Fibrosis Regional Centre of Florence (Italy), were investigated for they structural properties. Three strains of B. multivorans, three of B. cenocepacia and one of a Bcc member of undetermined genomovar were isolated from CF patients. The reference strains C1576 and J2315, for genomovar II and III, respectively, were included in the study. The bacteria were grown on solid media, the exopolysaccharides produced were purified, and their structures were determined. In addition, sugar analysis of sputum samples was accomplished to search for EPS produced in vivo. Six strains out of seven produced the exopolysaccharide cepacian, while one strain of B. multivorans produced a completely different polymer, previously known in the literature as PS1. Two strains synthesised very small amounts of EPS. No definitive evidence for the presence of cepacian in sputum samples was found. Most strains examined produced abundant amounts of polysaccharides. Cepacian was the most common EPS isolated and its production was not associated to a particular genomovar.

Outbreak of Corynebacterium pseudodiphtheriticum Infection in Cystic Fibrosis Patients, France

PubMed Central

Bittar, Fadi; Cassagne, Carole; Bosdure, Emmanuelle; Stremler, Nathalie; Dubus, Jean-Christophe; Sarles, Jacques; Reynaud-Gaubert, Martine; Raoult, Didier

2010-01-01

An increasing body of evidence indicates that nondiphtheria corynebacteria may be responsible for respiratory tract infections. We report an outbreak of Corynebacterium pseudodiphtheriticum infection in children with cystic fibrosis (CF). To identify 18 C. pseudodiphtheriticum strains isolated from 13 French children with CF, we used molecular methods (partial rpoB gene sequencing) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Clinical symptoms were exhibited by 10 children (76.9%), including cough, rhinitis, and lung exacerbations. The results of MALDI-TOF identification matched perfectly with those obtained from molecular identification. Retrospective analysis of sputum specimens by using specific real-time PCR showed that ≈20% of children with CF were colonized with these bacteria, whereas children who did not have CF had negative test results. Our study reemphasizes the conclusion that correctly identifying bacteria at the species level facilitates detection of an outbreak of new or emerging infections in humans. PMID:20678316

How are the ancient cystic fibrosis patients? Cystic fibrosis diagnosed over 60 years-old.

PubMed

Prados, C; Lerín, M; Cabanillas, J J; Gómez-Carrera, L; Álvarez-Sala, R

2017-01-01

To specify the prevalence of patients diagnosed with CF at age of ≥60 year-old and to analyze their characteristics. Observational study of CF patients which were diagnosed at age ≥60 year-old. The analyzed variables were: age, sex, nationality, lung function parameters, conditions present at diagnosis, microbiological characteristics and genetic findings. eight patients were included. 7 patients were female (87.5%) with a mean age of 70.6 years (median 71.5 years, range 60-78 years). The most important findings were: sweat test >60 mEq/l; heterozygotes F508del; bronchiectasis in CT; methicillin-sensitive Staphylococcus aureus (50%) in sputum. The most patients presented a normal or mild obstructive lung function. CF must also be considered a disease diagnosed in adulthood, incorporating the sweat test within the usual techniques of differential diagnosis in patients with different diseases associated with CF, because genetic counselling is esencial.

Best practices in the treatment of early cystic fibrosis lung disease.

PubMed

Proesmans, Marijke

2017-02-01

For many years, management of cystic fibrosis (CF) lung disease was focused on symptomatic treatment of chronic lung infection, which is characterized by cough and sputum production, leading to progressive lung damage. With increasing survival and better knowledge of the pathogenesis of CF lung disease, it has become clear that treatment has to start very early because lung damage occurs in young patients, often before obvious symptoms appear. The arrival of new cystic fibrosis transmembrane conductance-regulator (CFTR)-correcting therapies will bring more opportunities to prevent the disease, apart from only treating chronic lung infection. In this review, a summary of the current knowledge of early CF lung disease is provided, based on animal model studies, as well as on data obtained from well structured follow-up programs after newborn screening (NBS). The most important clinical guidelines for treating young CF patients are also summarized.

Sputum colour and bacteria in chronic bronchitis exacerbations: a pooled analysis.

PubMed

Miravitlles, Marc; Kruesmann, Frank; Haverstock, Daniel; Perroncel, Renee; Choudhri, Shurjeel H; Arvis, Pierre

2012-06-01

We examined the correlation between sputum colour and the presence of potentially pathogenic bacteria in acute exacerbations of chronic bronchitis (AECBs). Data were pooled from six multicentre studies comparing moxifloxacin with other antimicrobials in patients with an AECB. Sputum was collected before antimicrobial therapy, and bacteria were identified by culture and Gram staining. Association between sputum colour and bacteria was determined using logistic regression. Of 4,089 sputum samples, a colour was reported in 4,003; 1,898 (46.4%) were culture-positive. Green or yellow sputum samples were most likely to yield bacteria (58.9% and 45.5% of samples, respectively), compared with 18% of clear and 39% of rust-coloured samples positive for potentially pathogenic microorganisms. Factors predicting a positive culture were sputum colour (the strongest predictor), sputum purulence, increased dyspnoea, male sex and absence of fever. Green or yellow versus white sputum colour was associated with a sensitivity of 94.7% and a specificity of 15% for the presence of bacteria. Sputum colour, particularly green and yellow, was a stronger predictor of potentially pathogenic bacteria than sputum purulence and increased dyspnoea in AECB patients. However, it does not necessarily predict the need for antibiotic treatment in all patients with AECB.

Molecular Signature of Pseudomonas aeruginosa with Simultaneous Nanomolar Detection of Quorum Sensing Signaling Molecules at a Boron-Doped Diamond Electrode

NASA Astrophysics Data System (ADS)

Buzid, Alyah; Shang, Fengjun; Reen, F. Jerry; Muimhneacháin, Eoin Ó.; Clarke, Sarah L.; Zhou, Lin; Luong, John H. T.; O'Gara, Fergal; McGlacken, Gerard P.; Glennon, Jeremy D.

2016-07-01

Electroanalysis was performed using a boron-doped diamond (BDD) electrode for the simultaneous detection of 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline (HHQ) and pyocyanin (PYO). PQS and its precursor HHQ are two important signal molecules produced by Pseudomonas aeruginosa, while PYO is a redox active toxin involved in virulence and pathogenesis. This Gram-negative and opportunistic human pathogen is associated with a hospital-acquired infection particularly in patients with compromised immunity and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. Early detection is crucial in the clinical management of this pathogen, with established infections entering a biofilm lifestyle that is refractory to conventional antibiotic therapies. Herein, a detection procedure was optimized and proven for the simultaneous detection of PYO, HHQ and PQS in standard mixtures, biological samples, and P. aeruginosa spiked CF sputum samples with remarkable sensitivity, down to nanomolar levels. Differential pulse voltammetry (DPV) scans were also applicable for monitoring the production of PYO, HHQ and PQS in P. aeruginosa PA14 over 8 h of cultivation. The simultaneous detection of these three compounds represents a molecular signature specific to this pathogen.

Airway mucosal bioelectric potential difference in cystic fibrosis after lung transplantation.

PubMed

Wood, A; Higenbottam, T; Jackson, M; Scott, J; Stewart, S; Wallwork, J

1989-12-01

Bioelectrical potential difference (PD) across the respiratory mucosa is raised in cystic fibrosis (CF). We have recorded airway potentials from seven patients with CF who had undergone heart-lung transplantation and from eight patients without CF who had had transplants for cardiovascular disease; comparison of these populations controls for the effects of denervation and immunosuppressive treatment. Six patients without CF who had not had transplants formed an additional control. PD was recorded during routine fiberoptic bronchoscopy, using a Ringer's-perfused exploring bridge connected across a high impedance amplifier to an intravenous reference bridge. Bronchial lavage and sputum culture revealed no evidence of infection. Bronchial PD was similar in all three groups of patients at equivalent sites. However, nasal PD was raised in the CF group (mean value, 44 mV +/- 3.9 SE) compared with the patients who had transplants for cardiovascular disease (mean, 18 mV +/- 1.1 SE), and the control patients (mean, 15 mV +/- 1.2 SE). We conclude that the epithelial defects that result in raised airway potentials in CF do not recur in the transplanted lung.

Bolus Weekly Vitamin D3 Supplementation Impacts Gut and Airway Microbiota in Adults With Cystic Fibrosis: A Double-Blind, Randomized, Placebo-Controlled Clinical Trial.

PubMed

Kanhere, Mansi; He, Jiabei; Chassaing, Benoit; Ziegler, Thomas R; Alvarez, Jessica A; Ivie, Elizabeth A; Hao, Li; Hanfelt, John; Gewirtz, Andrew T; Tangpricha, Vin

2018-02-01

Disruption of gut microbiota may exacerbate severity of cystic fibrosis (CF). Vitamin D deficiency is a common comorbidity in patients with CF that may influence composition of the gut microbiota. Compare microbiota of vitamin D-sufficient and -insufficient CF patients and assess impact of a weekly high-dose vitamin D3 bolus regimen on gut and airway microbiome in adults with CF and vitamin D insufficiency (25-hydroxyvitamin D < 30 ng/mL). Forty-one subjects with CF were classified into two groups: vitamin D insufficient (n = 23) and vitamin D sufficient (n = 18). Subjects with vitamin D insufficiency were randomized to receive 50,000 IU of oral vitamin D3 or placebo weekly for 12 weeks. Sputum and stool samples were obtained pre- and postintervention and 16S ribosomal RNA genes sequenced using Illumina MiSeq technology. Gut microbiota differed significantly based on vitamin D status with Gammaproteobacteria, which contain numerous, potentially pathogenic species enriched in the vitamin D-insufficient group. Principal coordinates analysis showed differential gut microbiota composition within the vitamin D-insufficient patients following 12 weeks treatment with placebo or vitamin D3 (permutation multivariate analysis of variance = 0.024), with Lactococcus significantly enriched in subjects treated with vitamin D3, whereas Veillonella and Erysipelotrichaceae were significantly enriched in patients treated with placebo. This exploratory study suggests that vitamin D insufficiency is associated with alterations in microbiota composition that may promote inflammation and that supplementation with vitamin D has the potential to impact microbiota composition. Additional studies to determine the impact of vitamin D on microbiota benefit clinical outcomes in CF are warranted. Copyright © 2017 Endocrine Society

Incidence and clinical impact of respiratory viruses in adults with cystic fibrosis.

PubMed

Flight, William G; Bright-Thomas, Rowland J; Tilston, Peter; Mutton, Kenneth J; Guiver, Malcolm; Morris, Julie; Webb, A Kevin; Jones, Andrew M

2014-03-01

Viral respiratory infection (VRI) is a common cause of pulmonary exacerbations in children with cystic fibrosis (CF). The importance of VRI in adult CF populations is unclear. To determine the incidence and clinical impact of VRI among adults with CF. One hundred adults with CF were followed up prospectively for 12 months. Sputum, nose swabs and throat swabs were collected every 2 months and at onset of pulmonary exacerbation. PCR assays for adenovirus, influenza A&B, human metapneumovirus, parainfluenza 1-3, respiratory syncytial virus and human rhinovirus were performed on each sample. Symptom scores, spirometry and inflammatory markers were measured at each visit. One or more respiratory viruses were detected in 191/626 (30.5%) visits. Human rhinovirus accounted for 72.5% of viruses. Overall incidence of VRI was 1.66 (95% CI 1.39 to 1.92) cases/patient-year. VRI was associated with increased risk of pulmonary exacerbation (OR=2.19; 95% CI 1.56 to 3.08; p<0.001) and prescription of antibiotics (OR=2.26; 95% CI 1.63 to 3.13; p<0.001). Virus-positive visits were associated with higher respiratory symptom scores and greater C-reactive protein levels. Virus-positive exacerbations had a lower acute fall in FEV1 than virus-negative exacerbations (12.7% vs 15.6%; p=0.040). The incidence of exacerbations, but not VRI, was associated with greater lung function decline over 12 months (-1.79% per pulmonary exacerbation/year; 95% CI -3.4 to -0.23; p=0.025). VRI is common in adults with CF and is associated with substantial morbidity. Respiratory viruses are a potential therapeutic target in CF lung disease.

Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung.

PubMed

Kirchner, Sebastian; Fothergill, Joanne L; Wright, Elli A; James, Chloe E; Mowat, Eilidh; Winstanley, Craig

2012-06-05

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Giant African Pouched Rats as Detectors of Tuberculosis in Human Sputum: Comparison of Two Techniques for Sputum Presentation

ERIC Educational Resources Information Center

Wray, Alisha M.; Mahoney, Amanda; Weetjens, Bart J.; Cox, Christophe; Jubitana, Maureen; Kazwala, Rudovic; Mfinanga, Godfrey S.; Durgin, Amy; Poling, Alan

2013-01-01

Previous studies have shown that pouched rats can detect the presence of "Mycobacterium tuberculosis," which causes tuberculosis, in human sputum samples obtained from clinical facilities. Although pouched rats evaluate sputum samples quickly, preparing the samples is relatively slow. The present study evaluated whether the rats can detect…

Effects of inhaled high-molecular weight hyaluronan in inflammatory airway disease.

PubMed

Lamas, Adelaida; Marshburn, Jamie; Stober, Vandy P; Donaldson, Scott H; Garantziotis, Stavros

2016-10-03

Cystic fibrosis (CF) is a chronic inflammatory disease that is affecting thousands of patients worldwide. Adjuvant anti-inflammatory treatment is an important component of cystic fibrosis treatment, and has shown promise in preserving lung function and prolonging life expectancy. Inhaled high molecular weight hyaluronan (HMW-HA) is reported to improve tolerability of hypertonic saline and thus increase compliance, and has been approved in some European countries for use as an adjunct to hypertonic saline treatment in cystic fibrosis. However, there are theoretical concerns that HMW-HA breakdown products may be pro-inflammatory. In this clinical pilot study we show that sputum cytokines in CF patients receiving HMW-HA are not increased, and therefore HMW-HA does not appear to adversely affect inflammatory status in CF airways.

Purification and Characterization of a Mycelial Catalase from Scedosporium boydii, a Useful Tool for Specific Antibody Detection in Patients with Cystic Fibrosis

PubMed Central

Mina, Sara; Cimon, Bernard; Larcher, Gérald; Bouchara, Jean-Philippe; Robert, Raymond

2014-01-01

Scedosporium boydii is an opportunistic filamentous fungus which may be responsible for a wide variety of infections in immunocompetent and immunocompromised individuals. This fungus belongs to the Scedosporium apiospermum species complex, which usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF) and may lead to allergic bronchopulmonary mycoses, sensitization, or respiratory infections. Upon microbial infection, host phagocytic cells release reactive oxygen species (ROS), such as hydrogen peroxide, as part of the antimicrobial response. Catalases are known to protect pathogens against ROS by detoxification of the hydrogen peroxide. Here, we investigated the catalase equipment of Scedosporium boydii, one of the major pathogenic species in the S. apiospermum species complex. Three catalases were identified, and the mycelial catalase A1 was purified to homogeneity by a three-step chromatographic process. This enzyme is a monofunctional tetrameric protein of 460 kDa, consisting of four 82-kDa glycosylated subunits. The potential usefulness of this enzyme in serodiagnosis of S. apiospermum infections was then investigated by an enzyme-linked immunosorbent assay (ELISA), using 64 serum samples from CF patients. Whatever the species involved in the S. apiospermum complex, sera from infected patients were clearly differentiated from sera from patients with an Aspergillus fumigatus infection or those from CF patients without clinical and biological signs of a fungal infection and without any fungus recovered from sputum samples. These results suggest that catalase A1 is a good candidate for the development of an immunoassay for serodiagnosis of infections caused by the S. apiospermum complex in patients with CF. PMID:25355796

Purification and characterization of a mycelial catalase from Scedosporium boydii, a useful tool for specific antibody detection in patients with cystic fibrosis.

PubMed

Mina, Sara; Marot-Leblond, Agnès; Cimon, Bernard; Fleury, Maxime J J; Larcher, Gérald; Bouchara, Jean-Philippe; Robert, Raymond

2015-01-01

Scedosporium boydii is an opportunistic filamentous fungus which may be responsible for a wide variety of infections in immunocompetent and immunocompromised individuals. This fungus belongs to the Scedosporium apiospermum species complex, which usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF) and may lead to allergic bronchopulmonary mycoses, sensitization, or respiratory infections. Upon microbial infection, host phagocytic cells release reactive oxygen species (ROS), such as hydrogen peroxide, as part of the antimicrobial response. Catalases are known to protect pathogens against ROS by detoxification of the hydrogen peroxide. Here, we investigated the catalase equipment of Scedosporium boydii, one of the major pathogenic species in the S. apiospermum species complex. Three catalases were identified, and the mycelial catalase A1 was purified to homogeneity by a three-step chromatographic process. This enzyme is a monofunctional tetrameric protein of 460 kDa, consisting of four 82-kDa glycosylated subunits. The potential usefulness of this enzyme in serodiagnosis of S. apiospermum infections was then investigated by an enzyme-linked immunosorbent assay (ELISA), using 64 serum samples from CF patients. Whatever the species involved in the S. apiospermum complex, sera from infected patients were clearly differentiated from sera from patients with an Aspergillus fumigatus infection or those from CF patients without clinical and biological signs of a fungal infection and without any fungus recovered from sputum samples. These results suggest that catalase A1 is a good candidate for the development of an immunoassay for serodiagnosis of infections caused by the S. apiospermum complex in patients with CF. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Detection of Respiratory Viruses in Sputum from Adults by Use of Automated Multiplex PCR

PubMed Central

Walsh, Edward E.; Formica, Maria A.; Falsey, Ann R.

2014-01-01

Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 μL of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on “dunked” sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone. PMID:25056335

Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome.

PubMed

Klare, William; Das, Theerthankar; Ibugo, Amaye; Buckle, Edwina; Manefield, Mike; Manos, Jim

2016-08-01

Pseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome

PubMed Central

Das, Theerthankar; Ibugo, Amaye; Buckle, Edwina; Manefield, Mike; Manos, Jim

2016-01-01

Pseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies. PMID:27161630

Efficacy and Safety of Inhaled Aztreonam Lysine for Airway Pseudomonas in Cystic Fibrosis

PubMed Central

Retsch-Bogart, George Z.; Quittner, Alexandra L.; Gibson, Ronald L.; Oermann, Christopher M.; McCoy, Karen S.; Montgomery, A. Bruce; Cooper, Peter J.

2009-01-01

Background: We assessed the short-term efficacy and safety of aztreonam lysine for inhalation (AZLI [an aerosolized monobactam antibiotic]) in patients with cystic fibrosis (CF) and Pseudomonas aeruginosa (PA) airway infection. Methods: In this randomized, double-blind, placebo-controlled, international study (AIR-CF1 trial; June 2005 to April 2007), patients (n = 164; ≥ 6 years of age) with FEV1 ≥ 25% and ≤ 75% predicted values, and no recent use of antipseudomonal antibiotics or azithromycin were treated with 75 mg of AZLI (three times daily for 28 days) or placebo (1:1 randomization), then were monitored for 14 days after study drug completion. The primary efficacy end point was change in patient-reported respiratory symptoms (CF-Questionnaire-Revised [CFQ-R] Respiratory Scale). Secondary end points included changes in pulmonary function (FEV1), sputum PA density, and nonrespiratory CFQ-R scales. Adverse events and minimum inhibitory concentrations of aztreonam for PA were monitored. Results: After 28 days of treatment, AZLI improved the mean CFQ-R respiratory score (9.7 points; p < 0.001), FEV1 (10.3% predicted; p < 0.001), and sputum PA density (− 1.453 log10 cfu/g; p < 0.001), compared with placebo. Significant improvements in Eating, Emotional Functioning, Health Perceptions, Physical Functioning, Role Limitation/School Performance, and Vitality CFQ-R scales were observed. Adverse events were consistent with symptoms of CF lung disease and were comparable for AZLI and placebo except the incidence of “productive cough” was reduced by half in AZLI-treated patients. PA aztreonam susceptibility at baseline and end of therapy were similar. Conclusions: In patients with CF, PA airway infection, moderate-to-severe lung disease, and no recent use of antipseudomonal antibiotics or azithromycin, 28-day treatment with AZLI significantly improved respiratory symptoms and pulmonary function, and was well tolerated. Trial registration: Clinicaltrials.gov Identifier: NCT00112359 PMID:19420195

Association between spirometry controlled chest CT scores using computer-animated biofeedback and clinical markers of lung disease in children with cystic fibrosis.

PubMed

Kongstad, Thomas; Green, Kent; Buchvald, Frederik; Skov, Marianne; Pressler, Tania; Nielsen, Kim Gjerum

2017-01-01

Background : Computed tomography (CT) of the lungs is the gold standard for assessing the extent of structural changes in the lungs. Spirometry-controlled chest CT (SCCCT) has improved the usefulness of CT by standardising inspiratory and expiratory lung volumes during imaging. This was a single-centre cross-sectional study in children with cystic fibrosis (CF). Using SCCCT we wished to investigate the association between the quantity and extent of structural lung changes and pulmonary function outcomes, and prevalence of known CF lung pathogens. Methods : CT images were analysed by CF-CT scoring (expressed as % of maximum score) to quantify different aspects of structural lung changes including bronchiectasis, airway wall thickening, mucus plugging, opacities, cysts, bullae and gas trapping. Clinical markers consisted of outcomes from pulmonary function tests, microbiological cultures from sputum and serological samples reflecting anti-bacterial and anti-fungal antibodies. Results : Sixty-four children with CF, median age (range) of 12.7 (6.4-18.1) years, participated in the study. The median (range) CF-CT total score in all children was 9.3% (0.4-46.8) with gas trapping of 40.7% (3.7-100) as the most abundant finding. Significantly higher median CF-CT total scores (21.9%) were found in patients with chronic infections ( N  = 12) including Gram-negative infection and allergic bronchopulmonary aspergillosis (ABPA) exhibiting CF-CT total scores of 14.2% (ns) and 24.0% ( p  < 0.01), respectively, compared to 8.0% in patients with no chronic lung infection. Lung clearance index (LCI) derived from multiple breath washout exhibited closest association with total CF-CT scores, compared to other pulmonary function outcomes. Conclusions : The most prominent structural lung change was gas trapping, while CF-CT total scores were generally low, both showing close association with LCI. Chronic lung infections, specifically in the form of ABPA, were associated with increased scores in lung changes. Further investigation of impact of infections with different microorganisms on extent and progression of structural CF lung disease is needed.

[Molecular characterization of pathogenic bacteria of the respiratory tract in peruvian patients with cystic fibrosis].

PubMed

Aquino, Ruth; Gonzáles, Emely; Samaniego, Sol; Rivera, Juan; Cedeño, Virna; Urbina, Yrene; Diringer, Benoit

2017-01-01

To molecularly characterize the pathogenic bacteria of the respiratory tract isolated from patients with cystic fibrosis (CF) in Peru. Bacterial communities cultured from sputum samples of pediatric and adult patients with CF admitted to the Edgardo Rebagliati Martins National Hospital and the National Institute of Child Health were characterized. Standard microbiological techniques were used for bacterial culture, and gene sequencing of 16S rRNA and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and tandem MALDI-TOF mass spectrometry (MALDI TOF/TOF) were used for molecular characterization. Seventeen bacterial strains were characterized by 16S rRNA sequencing, and the identified pathogenic bacteria were Pseudomonas aeruginosa (31.5%), Staphylococcus aureus (12.6%), Pseudomonas spp. (11.8%), and Klebsiella oxytoca (3.1%). MALDI-TOF analysis generated a series of spectra representative of each isolated bacterial species, whereas MALDI TOF/TOF analysis identified the peptides and proteins of the most common strains and provided data on pathogenicity and sensitivity to antibiotics. The primary pathogenic microorganisms found in the respiratory tract of patients with CF in Peru were the same as those found in other countries. This study is the first to perform 16S rRNA sequencing as well as MALDI-TOF and MALDI-TOF/TOF analysis of the bacterial pathogens circulating in Peru. The inclusion of proteomic analysis further allowed for the identification of native microorganisms involved in CF.

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.

PubMed

Sung, Heungsup; Yong, Dongeun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na

2016-09-01

Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR

PubMed Central

Yong, Dongeun; Ki, Chang-Seok; Kim, Jae-Seok; Seong, Moon-Woo; Lee, Hyukmin

2016-01-01

Background Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. Methods We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). Results While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1–35.4 with the PK-DNase method, 34.7–39.0 with the PBS method, and 33.9–38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). Conclusions The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction. PMID:27374711

Development of Levofloxacin inhalation solution to treat Pseudomonas aeruginosa in patients with cystic fibrosis

PubMed Central

Stockmann, Chris; Sherwin, Catherine M.T.; Ampofo, Krow; Spigarelli, Michael G.

2017-01-01

Inhaled therapies allow for the targeted delivery of antimicrobials directly into the lungs and have been widely used in the treatment of cystic fibrosis (CF) acute pulmonary exacerbations. Nebulized levofloxacin solution (MP-376) is a novel therapy that is currently being evaluated in phase I, II, and III clinical trials among patients with stable CF and recent isolation of Pseudomonas aeruginosa from sputum. Phase I studies have investigated the single and multiple-dose pharmacokinetics of MP-376 and shown that it is rapidly absorbed from the lungs and results in low systemic concentrations. A subsequent phase IB study found that MP-376 pharmacokinetics were comparable among adults and children 6–16 years of age. Further phase II studies reported that sputum P. aeruginosa density decreased in a dose-dependent manner among patients who were randomized to MP-376 when compared with patients who received placebo. Improvements in pulmonary function and a decrease in the need for other antipseudomonal antibiotics were also reported for patients who received inhaled levofloxacin. The most common adverse event was dysgeusia (abnormal taste sensation), which was reported by nearly half of the participants who received MP-376. No serious drug-related adverse events were reported. These findings are encouraging; however, data from the two ongoing phase III trials are needed to determine whether MP-376 demonstrates substantial evidence of safety and efficacy as a chronic CF maintenance therapy and therefore may be useful in routine clinical practice. PMID:24334337

Population Structure, Antimicrobial Resistance, and Mutation Frequencies of Streptococcus pneumoniae Isolates from Cystic Fibrosis Patients

PubMed Central

del Campo, Rosa; Morosini, María-Isabel; de la Pedrosa, Elia Gómez-G.; Fenoll, Asunción; Muñoz-Almagro, Carmen; Máiz, Luis; Baquero, Fernando; Cantón, Rafael

2005-01-01

Forty-eight Streptococcus pneumoniae isolates recovered from sputum samples from 26 cystic fibrosis (CF) patients attending our CF unit (1995 to 2003) were studied. Mean yearly incidence of isolation was 5.5%, and all were strains recovered from young patients (≤12 years). The isolation was linked to clinical exacerbation in 35% of the cases, but only 27% of these were not accompanied by other CF pathogens. Fifty percent of the patients presented with two to four isolates over the studied period. Pulsed-field gel electrophoresis-SmaI digestion revealed a high heterogeneity (32 pulsotypes among 48 isolates) and the persistence over a 6-month period of a single clone (clone A) in two patients. This clone, presenting a varied multiresistance phenotype, was identified as the Spain23F-1 clone and was also recognized in six other patients, including two out of nine patients from the CF unit of Sant Joan de Dèu Hospital, Barcelona, Spain. In our isolates, 16 different serotypes were recognized, the most frequent being 23F (33.3%), 19F (18.8%), 6A (6.2%), and 6B (6.2%). High overall resistance rates were observed: to penicillin, 73%; to cefotaxime, 33%; to erythromycin, 42%; to tetracycline, 58%; to chloramphenicol, 48%; and to trimethoprim-sulfamethoxazole, 67%. Resistance to fluoroquinolones was not detected. Multiresistance was a common feature (60%). The percentage of S. pneumoniae strains with increased frequencies of mutation to rifampin resistance (≥7.5 × 10−8) was significantly higher (P = 0.02) in CF (60%) than among non-CF (37%) isolates in the same institution (M. I. Morosini et al., Antimicrob. Agents Chemother. 47:1464-1467, 2003). Even though a clear association with acute exacerbations could not be observed, long-term clonal persistence and variability, high frequency of antibiotic resistance, and hypermutability indicate the plasticity for adaptation of S. pneumoniae to the CF lung environment. PMID:15872243

Repurposing tromethamine as inhaled therapy to treat CF airway disease

PubMed Central

Alaiwa, Mahmoud H. Abou; Launspach, Janice L.; Sheets, Kelsey A.; Rivera, Jade A.; Gansemer, Nicholas D.; Taft, Peter J.; Thorne, Peter S.; Welsh, Michael J.; Stoltz, David A.

2016-01-01

In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR) anion channel activity causes airway surface liquid (ASL) pH to become acidic, which impairs airway host defenses. One potential therapeutic approach is to correct the acidic pH in CF airways by aerosolizing HCO3– and/or nonbicarbonate pH buffers. Here, we show that raising ASL pH with inhaled HCO3– increased pH. However, the effect was transient, and pH returned to baseline values within 30 minutes. Tromethamine (Tham) is a buffer with a long serum half-life used as an i.v. formulation to treat metabolic acidosis. We found that Tham aerosols increased ASL pH in vivo for at least 2 hours and enhanced bacterial killing. Inhaled hypertonic saline (7% NaCl) is delivered to people with CF in an attempt to promote mucus clearance. Because an increased ionic strength inhibits ASL antimicrobial factors, we added Tham to hypertonic saline and applied it to CF sputum. We found that Tham alone and in combination with hypertonic saline increased pH and enhanced bacterial killing. These findings suggest that aerosolizing the HCO3–-independent buffer Tham, either alone or in combination with hypertonic saline, might be of therapeutic benefit in CF airway disease. PMID:27390778

Molecular Signature of Pseudomonas aeruginosa with Simultaneous Nanomolar Detection of Quorum Sensing Signaling Molecules at a Boron-Doped Diamond Electrode

PubMed Central

Buzid, Alyah; Shang, Fengjun; Reen, F. Jerry; Muimhneacháin, Eoin Ó; Clarke, Sarah L.; Zhou, Lin; Luong, John H. T.; O’Gara, Fergal; McGlacken, Gerard P.; Glennon, Jeremy D.

2016-01-01

Electroanalysis was performed using a boron-doped diamond (BDD) electrode for the simultaneous detection of 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline (HHQ) and pyocyanin (PYO). PQS and its precursor HHQ are two important signal molecules produced by Pseudomonas aeruginosa, while PYO is a redox active toxin involved in virulence and pathogenesis. This Gram-negative and opportunistic human pathogen is associated with a hospital-acquired infection particularly in patients with compromised immunity and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. Early detection is crucial in the clinical management of this pathogen, with established infections entering a biofilm lifestyle that is refractory to conventional antibiotic therapies. Herein, a detection procedure was optimized and proven for the simultaneous detection of PYO, HHQ and PQS in standard mixtures, biological samples, and P. aeruginosa spiked CF sputum samples with remarkable sensitivity, down to nanomolar levels. Differential pulse voltammetry (DPV) scans were also applicable for monitoring the production of PYO, HHQ and PQS in P. aeruginosa PA14 over 8 h of cultivation. The simultaneous detection of these three compounds represents a molecular signature specific to this pathogen. PMID:27427496

Detection of Airway Colonization by Aspergillus fumigatus by Use of Electronic Nose Technology in Patients with Cystic Fibrosis.

PubMed

de Heer, K; Kok, M G M; Fens, N; Weersink, E J M; Zwinderman, A H; van der Schee, M P C; Visser, C E; van Oers, M H J; Sterk, P J

2016-03-01

Currently, there is no noninvasive test that can reliably diagnose early invasive pulmonary aspergillosis (IA). An electronic nose (eNose) can discriminate various lung diseases through an analysis of exhaled volatile organic compounds. We recently published a proof-of-principle study showing that patients with prolonged chemotherapy-induced neutropenia and IA have a distinct exhaled breath profile (or breathprint) that can be discriminated with an eNose. An eNose is cheap and noninvasive, and it yields results within minutes. We determined whether Aspergillus fumigatus colonization may also be detected with an eNose in cystic fibrosis (CF) patients. Exhaled breath samples of 27 CF patients were analyzed with a Cyranose 320. Culture of sputum samples defined the A. fumigatus colonization status. eNose data were classified using canonical discriminant analysis after principal component reduction. Our primary outcome was cross-validated accuracy, defined as the percentage of correctly classified subjects using the leave-one-out method. The P value was calculated by the generation of 100,000 random alternative classifications. Nine of the 27 subjects were colonized by A. fumigatus. In total, 3 subjects were misclassified, resulting in a cross-validated accuracy of the Cyranose detecting IA of 89% (P = 0.004; sensitivity, 78%; specificity, 94%). Receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.89. The results indicate that A. fumigatus colonization leads to a distinctive breathprint in CF patients. The present proof-of-concept data merit external validation and monitoring studies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sputum color: potential implications for clinical practice.

PubMed

Johnson, Allen L; Hampson, David F; Hampson, Neil B

2008-04-01

Respiratory infections with sputum production are a major reason for physician visits, diagnostic testing, and antibiotic prescription in the United States. We sought to determine whether the simple characteristic of sputum color provides information that impacts resource utilization such as laboratory testing and prescription of antibiotics. Out-patient sputum samples submitted to the microbiology laboratory for routine analysis were assigned to one of 8 color categories (green, yellow-green, rust, yellow, red, cream, white, and clear), based on a key made from paint chip color samples. Subsequent Gram stain and culture results were compared to sputum color. Of 289 consecutive samples, 144 (50%) met standard Gram-stain criteria for being acceptable lower-respiratory-tract specimens. In the acceptable Gram-stain group, 60 samples had a predominant organism on Gram stain, and the culture yielded a consistent result in 42 samples (15% of the 289 total specimens). Yield at each level of analysis differed greatly by color. The yield from sputum colors green, yellow-green, yellow, and rust was much higher than the yield from cream, white, or clear. If out-patient sputum is cream, white, or clear, the yield from bacteriologic analysis is extremely low. This information can reduce laboratory processing costs and help minimize unnecessary antibiotic prescription.

Freezing of homogenized sputum samples for intermittent storage.

PubMed

Holz, O; Mücke, M; Zarza, P; Loppow, D; Jörres, R A; Magnussen, H

2001-08-01

Among the reasons that restrict the application of sputum induction in outpatient settings is the need for processing of samples within 2 h after induction. The aim of our study was to assess whether freezing is suitable for intermediate storage of sputum samples before processing. We compared differential cell counts between two sputum aliquots derived from the same sample. One aliquot was processed within 2 h after production and one, after it had been frozen under addition of dimethyl-sulfoxid (DMSO) and stored up to 10 days at -20 degrees C. Thirty-five samples were frozen immediately prior to preparation of cytospins, and 10 samples were frozen at an even earlier stage, directly after homogenization. In both sets of experiments we observed a significant relationship between frozen and native samples regarding macrophages, neutrophils and eosinophils, as indicated by respective intraclass correlation coefficients of 0.96, 0.96, and 0.93 in the first, and of 0.92, 0.96 and 0.77 in the second experiments. Our results indicate that the freezing of sputum samples at different stages of processing does not alter sputum morphology to an extent that affects the results of differential cell counts.

Biofilms produced by Burkholderia cenocepacia: influence of media and solid supports on composition of matrix exopolysaccharides.

PubMed

Pellizzoni, Elena; Ravalico, Fabio; Scaini, Denis; Delneri, Ambra; Rizzo, Roberto; Cescutti, Paola

2016-02-01

Bacteria usually grow forming biofilms, which are communities of cells embedded in a self-produced dynamic polymeric matrix, characterized by a complex three-dimensional structure. The matrix holds cells together and above a surface, and eventually releases them, resulting in colonization of other surfaces. Although exopolysaccharides (EPOLs) are important components of the matrix, determination of their structure is usually performed on samples produced in non-biofilm conditions, or indirectly through genetic studies. Among the Burkholderia cepacia complex species, Burkholderia cenocepacia is an important pathogen in cystic fibrosis (CF) patients and is generally more aggressive than other species. In the present investigation, B. cenocepacia strain BTS2, a CF isolate, was grown in biofilm mode on glass slides and cellulose membranes, using five growth media, one of which mimics the nutritional content of CF sputum. The structure of the matrix EPOLs was determined by 1H-NMR spectroscopy, while visualization of the biofilms on glass slides was obtained by means of confocal laser microscopy, phase-contrast microscopy and atomic force microscopy. The results confirmed that the type of EPOLs biosynthesized depends both on the medium used and on the type of support, and showed that mucoid conditions do not always lead to significant biofilm production, while bacteria in a non-mucoid state can still form biofilm containing EPOLs.

Evidence for a Cystic Fibrosis Enteropathy

PubMed Central

Adriaanse, Marlou P. M.; van der Sande, Linda J. T. M.; van den Neucker, Anita M.; Menheere, Paul P. C. A.; Dompeling, Edward; Buurman, Wim A.; Vreugdenhil, Anita C. E.

2015-01-01

Background Previous studies have suggested the existence of enteropathy in cystic fibrosis (CF), which may contribute to intestinal function impairment, a poor nutritional status and decline in lung function. This study evaluated enterocyte damage and intestinal inflammation in CF and studied its associations with nutritional status, CF-related morbidities such as impaired lung function and diabetes, and medication use. Methods Sixty-eight CF patients and 107 controls were studied. Levels of serum intestinal-fatty acid binding protein (I-FABP), a specific marker for enterocyte damage, were retrospectively determined. The faecal intestinal inflammation marker calprotectin was prospectively studied. Nutritional status, lung function (FEV1), exocrine pancreatic insufficiency (EPI), CF-related diabetes (CFRD) and use of proton pump inhibitors (PPI) were obtained from the medical charts. Results Serum I-FABP levels were elevated in CF patients as compared with controls (p<0.001), and correlated negatively with FEV1 predicted value in children (r-.734, p<0.05). Faecal calprotectin level was elevated in 93% of CF patients, and correlated negatively with FEV1 predicted value in adults (r-.484, p<0.05). No correlation was found between calprotectin levels in faeces and sputum. Faecal calprotectin level was significantly associated with the presence of CFRD, EPI, and PPI use. Conclusion This study demonstrated enterocyte damage and intestinal inflammation in CF patients, and provides evidence for an inverse correlation between enteropathy and lung function. The presented associations of enteropathy with important CF-related morbidities further emphasize the clinical relevance. PMID:26484665

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease

PubMed Central

Bacci, Giovanni; Fiscarelli, Ersilia; Taccetti, Giovanni; Dolce, Daniela; Paganin, Patrizia; Morelli, Patrizia; Tuccio, Vanessa; De Alessandri, Alessandra; Lucidi, Vincenzina

2017-01-01

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease. PMID:28758937

Metagenomic Analysis of Respiratory Tract DNA Viral Communities in Cystic Fibrosis and Non-Cystic Fibrosis Individuals

PubMed Central

Haynes, Matthew; Schmieder, Robert; Angly, Florent E.; Silva, Joas; Tammadoni, Sassan; Nosrat, Bahador; Conrad, Douglas; Rohwer, Forest

2009-01-01

The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF) and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5′-triphosphate,3′-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota. PMID:19816605

Effect of oral glycine on the clinical, spirometric and inflammatory status in subjects with cystic fibrosis: a pilot randomized trial.

PubMed

Vargas, Mario H; Del-Razo-Rodríguez, Rosangela; López-García, Amando; Lezana-Fernández, José Luis; Chávez, Jaime; Furuya, María E Y; Marín-Santana, Juan Carlos

2017-12-15

Patients with cystic fibrosis (CF) have airway inflammation that contributes to symptoms and to pulmonary function derangement. Current drugs used to diminish airway inflammation improve the clinical and spirometric status of patients with CF, but their use is limited due to their undesired side effects, for example, glucose intolerance, growth retardation, and cataracts with corticosteroids, gastrointestinal toxicity with ibuprofen, and macrolide resistance with azythromycin. Glycine is known to decrease activation of inflammatory cells, including alveolar macrophages and neutrophils, and is relatively inexpensive, palatable, and virtually devoid of untoward effects. These features make glycine a good candidate for antiinflammatory treatment of CF. Thus, we aimed to explore whether glycine can exert a beneficial effect in a population of patients with CF. This was a randomized, double blinded, cross-over pilot clinical trial. Subjects with CF received, in random order, oral glycine (0.5 g/kg/day, dissolved in any liquid) and placebo (glass sugar), each during 8 weeks with an intermediate 2-week wash-out period. Thirteen subjects aged 6-23 years, 8 females, completed the two arms of the study. As compared with placebo, after glycine intake patients had better symptom questionnaire scores (p = 0.02), mainly regarding sputum features and dyspnea. While spirometric variables tended to decline during placebo intake, they remained stable or even increased during glycine treatment (p = 0.04 to p = 0.003). In this context, FEV 1 declined 8.6% after placebo and increased 9.7% at the end of the glycine period. Pulse oximetry improved after glycine intake (p = 0.04 vs. placebo). TNF-α in serum and IL-6 and G-CSF in sputum tended to decline at the end of the glycine period (p = 0.061, p = 0.068 and p = 0.04, respectively, vs placebo). Glycine was remarkably well tolerated. The clinical, spirometric and inflammatory status of subjects with CF improved after just 8 weeks of glycine intake, suggesting that this amino acid might constitute a novel therapeutic tool for these patients. Thus, further studies are warranted. www.clinicaltrials.gov , registration number: NCT01417481 , date of registration: March 12, 2012.

[Management of infants whose diagnosis is inconclusive at neonatal screening for cystic fibrosis].

PubMed

Sermet-Gaudelus, I; Brouard, J; Audrézet, M-P; Couderc Kohen, L; Weiss, L; Wizla, N; Vrielynck, S; LLerena, K; Le Bourgeois, M; Deneuville, E; Remus, N; Nguyen-Khoa, T; Raynal, C; Roussey, M; Girodon, E

2017-04-01

Neonatal screening for cystic fibrosis (CF) may detect infants with elevated immunoreactive trypsinogen (IRT) levels but with inconclusive sweat tests and/or DNA results. This includes cases associating (1) either the presence of at most one CF-causing mutation and sweat chloride values between 30 and 59mmol/L or (2) two CFTR mutations with at least one of unknown pathogenicity and a sweat chloride below 60mmol/L. This encompasses different clinical situations whose progression cannot be predicted. These cases require redoing the sweat test at 12 months and if possible at 6 and 24 months of life. This must be associated with extended genotyping. CFTR functional explorations can also help by investigating CFTR dysfunction. These infants must be initially evaluated in dedicated CF centers including bacteriological sputum analysis, chest radiology and fecal elastase dosage. A home practitioner must be informed of the specificity of follow-up. These infants will be reviewed in the CF center at 3, 6 and 12 months and every year. Any CF-related symptom requires reevaluation of the diagnosis. These guidelines were established by the "neonatal screening and difficult diagnoses" working group of the French CF Society. They aim to standardize management of infants with unclear diagnosis in French CF centers. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Synthetic cystic fibrosis sputum medium diminishes Burkholderia cenocepacia antifungal activity against Aspergillus fumigatus independently of phenylacetic acid production.

PubMed

Lightly, Tasia Joy; Phung, Ryan R; Sorensen, John L; Cardona, Silvia T

2017-05-01

Phenylacetic acid (PAA), an intermediate of phenylalanine degradation, is emerging as a signal molecule in microbial interactions with the host. In this work, we explore the presence of phenylalanine and PAA catabolism in 3 microbial pathogens of the cystic fibrosis (CF) lung microbiome: Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aspergillus fumigatus. While in silico analysis of B. cenocepacia J2315 and A. fumigatus Af293 genome sequences showed complete pathways from phenylalanine to PAA, the P. aeruginosa PAO1 genome lacked several coding genes for phenylalanine and PAA catabolic enzymes. High-performance liquid chromatography analysis of supernatants from B. cenocepacia K56-2 detected PAA when grown in Luria-Bertani medium but not in synthetic cystic fibrosis sputum medium (SCFM). However, we were unable to identify PAA production by A. fumigatus or P. aeruginosa in any of the conditions tested. The inhibitory effect of B. cenocepacia on A. fumigatus growth was evaluated using agar plate interaction assays. Inhibition of fungal growth by B. cenocepacia was lessened in SCFM but this effect was not dependent on bacterial production of PAA. In summary, while we demonstrated PAA production by B. cenocepacia, we were not able to link this metabolite with the B. cenocepacia - A. fumigatus microbial interaction in CF nutritional conditions.

Sputum colour for diagnosis of a bacterial infection in patients with acute cough.

PubMed

Altiner, Attila; Wilm, Stefan; Däubener, Walter; Bormann, Christiane; Pentzek, Michael; Abholz, Heinz-Harald; Scherer, Martin

2009-01-01

Sputum colour plays an important role in the disease concepts for acute cough, both in the patients' and the doctors' view. However, it is unclear whether the sputum colour can be used for diagnosis of a bacterial infection. Cross-sectional study. A total of 42 GP practices in Dusseldorf, Germany. Sputum samples obtained from 241 patients suffering from an episode of acute cough seeing their doctor within a routine consultation. Relation of sputum colour and microbiological proof of bacterial infection defined as positive culture and at least a moderate number of leucocytes per low magnification field. In 28 samples (12%) a bacterial infection was proven. Yellowish or greenish colour of the sputum sample and bacterial infection showed a significant correlation (p = 0.014, Fisher's exact test). The sensitivity of yellowish or greenish sputum used as a test for a bacterial infection was 0.79 (95% CI 0.63-0.94); the specificity was 0.46 (95% CI 0.038-0.53). The positive likelihood-ratio (+LR) was 1.46 (95% CI 1.17-1.85). The sputum colour of patients with acute cough and no underlying chronic lung disease does not imply therapeutic consequences such as prescription of antibiotics.

Evaluation of OMNIgene®•SPUTUM-stabilised sputum for long-term transport and Xpert® MTB/RIF testing in Nepal.

PubMed

Maharjan, B; Kelly-Cirino, C D; Weirich, A; Curry, P S; Hoffman, H; Avsar, K; Shrestha, B

2016-12-01

German Nepal TB Project, National Tuberculosis Reference Laboratory, Kathmandu, Nepal. To evaluate whether transporting samples in OMNIgene®•SPUTUM (OM-S) reagent from a peripheral collection site to a central laboratory in Nepal can improve tuberculosis (TB) detection and increase the sensitivity of Xpert® MTB/RIF testing. One hundred sputum samples were split manually. Each portion was assigned to the OM-S group (OM-S added at collection, airline-couriered without cold chain, no other processing required) or the standard-of-care (SOC) group (samples airline-couriered on ice, sodium hydroxide + N-acetyl-L-cysteine processing required at the laboratory). Smear microscopy and Xpert testing were performed. Transport time was 2-13 days. Overall smear results were comparable (respectively 58% and 56% smear-negative results in the OM-S and SOC groups). The rate of smear-positive, Mycobacterium tuberculosis-positive (MTB+) sample detection was identical for both treatment groups, at 95%. More smear-negative MTB+ samples were detected in the OM-S group (17% vs. 13%, P = 0.0655). Sputum samples treated with OM-S can undergo multiday ambient-temperature transport and yield comparable smear and Xpert results to those of SOC samples. Further investigation with larger sample sizes is required to assess whether treating sputum samples with OM-S could increase the sensitivity of Xpert testing in smear-negative samples.

Induced sputum and bronchoscopy in the diagnosis of pulmonary tuberculosis

PubMed Central

McWilliams, T; Wells, A; Harrison, A; Lindstrom, S; Cameron, R; Foskin, E

2002-01-01

Background: Previous studies suggest that bronchoscopy and a single induced sputum sample are equally effective for diagnosing pulmonary tuberculosis. Methods: In a prospective study of subjects with possibly active pulmonary tuberculosis, the diagnostic yield of three induced sputum tests was compared with that of bronchoscopy. Subjects either produced no sputum or (acid fast) smear negative sputum. Bronchoscopy was only performed if at least two induced sputum samples were smear negative. Results: Of 129 subjects who completed all tests, 27 (21%) had smear negative and culture positive specimens, 14 (52%) on bronchoscopy and 26 (96%) on induced sputum (p<0.005). One patient was culture positive on bronchoscopy alone compared with 13 on induced sputum alone; 13 were culture positive on both tests. Induced sputum positivity was strikingly more prevalent when chest radiographic appearances showed any features of active tuberculosis (20/63, 32%) than when appearances suggested inactivity (1/44, 2%; p<0.005). Induced sputum costs were about one third those of bronchoscopy, and the ratio of costs of the two tests per case of tuberculosis diagnosed could be as much as 1:6. Conclusions: In subjects investigated for possibly active or inactive tuberculosis who produce no sputum or have smear negative sputum, the most cost effective strategy is to perform three induced sputum tests without bronchoscopy. Induced sputum testing carries a high risk of nosocomial tuberculosis unless performed in respiratory isolation conditions. The cost benefits shown could be lost if risk management measures are not observed. PMID:12454293

Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.

PubMed

Héry-Arnaud, G; Nowak, E; Caillon, J; David, V; Dirou, A; Revert, K; Munck, M-R; Frachon, I; Haloun, A; Horeau-Langlard, D; Le Bihan, J; Danner-Boucher, I; Ramel, S; Pelletier, M-P; Rosec, S; Gouriou, S; Poulhazan, E; Payan, C; Férec, C; Rault, G; Le Gal, G; Le Berre, R

2017-03-01

Early detection of Pseudomonas aeruginosa lung positivity is a key element in cystic fibrosis (CF) management. PCR has increased the accuracy of detection of many microorganisms. Clinical relevance of P. aeruginosa quantitative PCR (qPCR) in this context is unclear. Our aim was to determine P. aeruginosa qPCR sensitivity and specificity, and to assess the possible time saved by qPCR in comparison with standard practice (culture). A multicentre cohort study was conducted over a 3-year period in 96 patients with CF without chronic P. aeruginosa colonization. Sputum samples were collected at each visit. Conventional culture and two-step qPCR (oprL qPCR and gyrB/ecfX qPCR) were performed for 707 samples. The positivity criteria were based on the qPCR results, defined in a previous study as follow: oprL qPCR positivity alone if bacterial density was <730 CFU/mL or oprL qPCR combined with gyrB/ecfX qPCR if bacterial density was ≥730 CFU/mL. During follow up, 36 of the 96 patients with CF were diagnosed on culture as colonized with P. aeruginosa. This two-step qPCR displayed a sensitivity of 94.3% (95% CI 79.7%-98.6%), and a specificity of 86.3% (95% CI 83.4%-88.7%). It enabled P. aeruginosa acquisition to be diagnosed earlier in 20 patients, providing a median detection time gain of 8 months (interquartile range 3.7-17.6) for them. Implementing oprL and gyrB/ecfX qPCR in the management of patients with CF allowed earlier detection of first P. aeruginosa lung positivity than culture alone. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Mannitol versus hypertonic saline: Safety and efficacy of mannitol and hypertonic saline in sputum induction and bronchial hyperreactivity assessment.

PubMed

Alvarez-Puebla, M J; Olaguibel, J M; Almudevar, E; Echegoyen, A A; Vela, C; de Esteban, B

2015-08-01

Eosinophilic asthma phenotype predicts good response to corticosteroids and associates to asthmatic exacerbations. Sputum induction by hypertonic saline (HS) inhalation is technically demanding. Bronchial hyperresponsiveness (BHR) to osmotic agents indirectly mirrors active airway inflammation. We compared the safety and ability of HS and mannitol to induce sputum and measure BHR. We evaluated the stability of inflammatory phenotypes. We studied 35 non-smoking asthmatics randomized to undergo HS and mannitol challenges on 2 days 1 week apart. Sputum was sampled for cell analysis and phenotyped as eosinophilic (≥3% eosinophils) and non-eosinophilic (<3%) asthma. Nineteen subjects had BHR to mannitol and nine of them also had BHR to HS. Drops in forced expiratory volume in 1 s were higher from HS challenge than from mannitol challenge. Adequate sputum samples were obtained from 80% subjects (68% mannitol and 71% HS). Eosinophils and macrophages from both challenges correlated. Neutrophils were higher in sputum from HS. Ninety percent samples were equally phenotyped with HS and mannitol. Fractional exhaled nitric oxide, sputum eosinophils and BHR correlated in both challenges. HS and mannitol showed similar capacity to produce valuable sputum samples. BHR to both osmotic stimuli partially resembled airway eosinophilic inflammation but mannitol was more sensitive than HS to assess BHR. Eosinophilic phenotype remained stable in most patients with both stimuli. © The Author(s) 2015.

Non-small cell lung cancer detection using microRNA expression profiling of bronchoalveolar lavage fluid and sputum.

PubMed

Kim, Julian O; Gazala, Sayf; Razzak, Rene; Guo, Linghong; Ghosh, Sunita; Roa, Wilson H; Bédard, Eric L R

2015-04-01

To assess if miRNA expression profiling of bronchoalveolar lavage (BAL) fluid and sputum could be used to detect early-stage non-small cell lung cancer (NSCLC). Hierarchical cluster analysis was performed on the expression levels of 5 miRNAs (miR-21, miR-143, miR-155, miR-210, and miR-372) which were quantified using RNA reverse transcription and quantitative real-time polymerase chain reaction in sputum and BAL samples from NSCLC cases and cancer-free controls. Cluster analysis of the miRNA expression levels in BAL samples from 21 NSCLC cases and sputum samples from 10 cancer-free controls yielded a diagnostic sensitivity of 85.7% and specificity of 100%. Cluster analysis of sputum samples from the same patients yielded a diagnostic sensitivity of 67.8% and specificity of 90%. miRNA expression profiling of sputum and BAL fluids represent a potential means to detect early-stage NSCLC. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The ferrous iron-responsive BqsRS two-component system activates genes that promote cationic stress tolerance.

PubMed

Kreamer, Naomi N; Costa, Flavia; Newman, Dianne K

2015-02-24

The physiological resistance of pathogens to antimicrobial treatment is a severe problem in the context of chronic infections. For example, the mucus-filled lungs of cystic fibrosis (CF) patients are readily colonized by diverse antibiotic-resistant microorganisms, including Pseudomonas aeruginosa. Previously, we showed that bioavailable ferrous iron [Fe(II)] is present in CF sputum at all stages of infection and constitutes a significant portion of the iron pool at advanced stages of lung function decline [R. C. Hunter et al., mBio 4(4):e00557-13, 2013]. P. aeruginosa, a dominant CF pathogen, senses Fe(II) using a two-component signal transduction system, BqsRS, which is transcriptionally active in CF sputum [R. C. Hunter et al., mBio 4(4):e00557-13, 2013; N. N. Kreamer, J. C. Wilks, J. J. Marlow, M. L. Coleman, and D. K. Newman, J Bacteriol 194:1195-1204, 2012]. Here, we show that an RExxE motif in BqsS is required for BqsRS activation. Once Fe(II) is sensed, BqsR binds a tandem repeat DNA sequence, activating transcription. The BqsR regulon--defined through iterative bioinformatic predictions and experimental validation--includes several genes whose products are known to drive antibiotic resistance to aminoglycosides and polymyxins. Among them are genes encoding predicted determinants of polyamine transport and biosynthesis. Compared to the wild type, bqsS and bqsR deletion mutants are sensitive to high levels of Fe(II), produce less spermidine in high Fe(II), and are more sensitive to tobramycin and polymyxin B but not arsenate, chromate, or cefsulodin. BqsRS thus mediates a physiological response to Fe(II) that guards the cell against positively charged molecules but not negatively charged stressors. These results suggest Fe(II) is an important environmental signal that, via BqsRS, bolsters tolerance of a variety of cationic stressors, including clinically important antimicrobial agents. Clearing chronic infections is challenging due to the physiological resistance of opportunistic pathogens to antibiotics. Effective treatments are hindered by a lack of understanding of how these organisms survive in situ. Fe(II) is typically present at micromolar levels in soils and sedimentary habitats, as well as in CF sputum. All P. aeruginosa strains possess a two-component system, BqsRS, that specifically senses extracellular Fe(II) at low micromolar concentrations. Our work shows that BqsRS protects the cell against cationic perturbations to the cell envelope as well as low pH and reduction potential (Eh), conditions under which Fe(2+) is stable. Fe(II) can thus be understood as a proxy for a broader environmental state; the cellular response to its detection may help rationalize the resistance of P. aeruginosa to clinically important cationic antibiotics. This finding demonstrates the importance of considering environmental chemistry when exploring mechanisms of microbial survival in habitats that include the human body. Copyright © 2015 Kreamer et al.

Sputum colour for diagnosis of a bacterial infection in patients with acute cough

PubMed Central

Altiner, Attila; Wilm, Stefan; Däubener, Walter; Bormann, Christiane; Pentzek, Michael; Abholz, Heinz-Harald; Scherer, Martin

2009-01-01

Objective Sputum colour plays an important role in the disease concepts for acute cough, both in the patients’ and the doctors’ view. However, it is unclear whether the sputum colour can be used for diagnosis of a bacterial infection. Design Cross-sectional study. Setting A total of 42 GP practices in Düsseldorf, Germany. Subjects Sputum samples obtained from 241 patients suffering from an episode of acute cough seeing their doctor within a routine consultation. Main outcome measures Relation of sputum colour and microbiological proof of bacterial infection defined as positive culture and at least a moderate number of leucocytes per low magnification field. Results In 28 samples (12%) a bacterial infection was proven. Yellowish or greenish colour of the sputum sample and bacterial infection showed a significant correlation (p = 0.014, Fisher's exact test). The sensitivity of yellowish or greenish sputum used as a test for a bacterial infection was 0.79 (95% CI 0.63–0.94); the specificity was 0.46 (95% CI 0.038–0.53). The positive likelihood-ratio (+LR) was 1.46 (95% CI 1.17-1.85). Conclusions The sputum colour of patients with acute cough and no underlying chronic lung disease does not imply therapeutic consequences such as prescription of antibiotics. PMID:19242860

Cohort Study of Airway Mycobiome in Adult Cystic Fibrosis Patients: Differences in Community Structure between Fungi and Bacteria Reveal Predominance of Transient Fungal Elements

PubMed Central

Sauer-Heilborn, Annette; Welte, Tobias; Guzman, Carlos A.; Abraham, Wolf-Rainer; Höfle, Manfred G.

2015-01-01

The respiratory mycobiome is an important but understudied component of the human microbiota. Like bacteria, fungi can cause severe lung diseases, but their infection rates are much lower. This study compared the bacterial and fungal communities of sputum samples from a large cohort of 56 adult patients with cystic fibrosis (CF) during nonexacerbation periods and under continuous antibiotic treatment. Molecular fingerprinting based on single-strand conformation polymorphism (SSCP) analysis revealed fundamental differences between bacterial and fungal communities. Both groups of microorganisms were taxonomically classified by identification of gene sequences (16S rRNA and internal transcript spacer), and prevalences of single taxa were determined for the entire cohort. Major bacterial pathogens were frequently observed, whereas fungi of known pathogenicity in CF were detected only in low numbers. Fungal species richness increased without reaching a constant level (saturation), whereas bacterial richness showed saturation after 50 patients were analyzed. In contrast to bacteria, a large number of fungal species were observed together with high fluctuations over time and among patients. These findings demonstrated that the mycobiome was dominated by transient species, which strongly suggested that the main driving force was their presence in inhaled air rather than colonization. Considering the high exposure of human airways to fungal spores, we concluded that fungi have low colonization abilities in CF, and colonization by pathogenic fungal species may be considered a rare event. A comprehensive understanding of the conditions promoting fungal colonization may offer the opportunity to prevent colonization and substantially reduce or even eliminate fungus-related disease progression in CF. PMID:26135861

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

PubMed

Coron, Noémie; Pihet, Marc; Fréalle, Emilie; Lemeille, Yolande; Pinel, Claudine; Pelloux, Hervé; Gargala, Gilles; Favennec, Loic; Accoceberry, Isabelle; Durand-Joly, Isabelle; Dalle, Frédéric; Huet, Frédéric; Fanton, Annlyse; Boldron, Amale; Loeuille, Guy-André; Domblides, Philippe; Coltey, Bérengère; Pin, Isabelle; Llerena, Catherine; Troussier, Françoise; Person, Christine; Marguet, Christophe; Wizla, Nathalie; Thumerelle, Caroline; Turck, Dominique; Bui, Stéphanie; Fayon, Michael; Duhamel, Alain; Prévotat, Anne; Wallaert, Benoit; Leroy, Sylvie; Bouchara, Jean-Philippe; Delhaes, Laurence

2018-02-01

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 3 weeks. 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 16 days 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.

Fate of sputum samples transported in bottles containing cetylpyridinium chloride and sodium chloride: a national reference laboratory study.

PubMed

Visalakshi, Potharaju; Meharwal, Sandeep K; Arora, Jyoti; Bhalla, Manpreet; Singh, Niti; Myneedu, Vithal Prasad; Behera, Digamber

2012-04-01

The transportation of sputum samples may sometimes take more than one week which results in an increased contamination rate and loss of positive cultures. The current study was planned to analyze the recovery rate of mycobacteria from transported samples with and without Cetylpyridinium chloride (CPC). Addition of CPC is useful for isolation of M. tuberculosis from sputum subjected to long-term storage.

Validation of sputum Gram stain for treatment of community-acquired pneumonia and healthcare-associated pneumonia: a prospective observational study.

PubMed

Fukuyama, Hajime; Yamashiro, Shin; Kinjo, Kiyoshi; Tamaki, Hitoshi; Kishaba, Tomoo

2014-10-18

The usefulness of sputum Gram stain in patients with community-acquired pneumonia (CAP) is controversial. There has been no study to evaluate the diagnostic value of this method in patients with healthcare-associated pneumonia (HCAP). The purpose of this study was to evaluate the usefulness of sputum Gram stain in etiological diagnosis and pathogen-targeted antibiotic treatment of CAP and HCAP. We conducted a prospective observational study on hospitalized patients with pneumonia admitted to our hospital from August 2010 to July 2012. Before administering antibiotics on admission, Gram stain was performed and examined by trained physicians immediately after sputum samples were obtained. We analyzed the quality of sputum samples and the diagnostic performance of Gram stain. We also compared pathogen-targeted antibiotic treatment guided by sputum Gram stain with empirical treatment. Of 670 patients with pneumonia, 328 were CAP and 342 were HCAP. Sputum samples were obtained from 591 patients, of these 478 samples were good quality. The sensitivity and specificity of sputum Gram stain were 62.5% and 91.5% for Streptococcus pneumoniae, 60.9% and 95.1% for Haemophilus influenzae, 68.2% and 96.1% for Moraxella catarrhalis, 39.5% and 98.2% for Klebsiella pneumoniae, 22.2% and 99.8% for Pseudomonas aeruginosa, 9.1% and 100% for Staphylococcus aureus. The diagnostic yield decreased in patients who had received antibiotics or patients with suspected aspiration pneumonia. Pathogen-targeted treatment provided similar efficacy with a decrease in adverse events compared to empirical treatment. Sputum Gram stain is highly specific for the etiologic diagnosis and useful in guiding pathogen-targeted antibiotic treatment of CAP and HCAP.

The fate of inhaled antibiotics after deposition in cystic fibrosis: How to get drug to the bug?

PubMed

Bos, Aukje C; Passé, Kimberly M; Mouton, Johan W; Janssens, Hettie M; Tiddens, Harm A W M

2017-01-01

Chronic airway infections in patients with cystic fibrosis (CF) are most often treated with inhaled antibiotics of which deposition patterns have been extensively studied. However, the journey of aerosol particles does not end after deposition within the bronchial tree. To review how local conditions affect the clinical efficacy of antibiotic aerosol particles after deposition in the airways of patients with CF. Electronic databases were searched from inception to September 2015. Original studies describing the effect of CF sputum or bacterial factors on antibiotic efficacy and formulations to increase efficacy were included. 35 articles were included which mostly described in vitro studies and mainly investigated aminoglycosides. After deposition, diffusion through the mucus layer was reduced for aminoglycosides, β-lactam antibiotics and fluoroquinolones. Within CF mucus, low oxygen tension adversely affected aminoglycosides, β-lactam antibiotics, and chloramphenicol; and molecules inactivated aminoglycosides but not β-lactam antibiotics. Finally, the alginate layer surrounding Pseudomonas aeruginosa was an important factor in the resistance against all antibiotics. After deposition in the airways, the local efficacy of inhaled antibiotics can be reduced by molecules within CF mucus and the alginate layer surrounding P. aeruginosa. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Trampoline use as physiotherapy for cystic fibrosis patients.

PubMed

Barak, A; Wexler, I D; Efrati, O; Bentur, L; Augarten, A; Mussaffi, H; Avital, A; Rivlin, J; Aviram, M; Yahav, Y; Kerem, E

2005-01-01

Physicians and physiotherapists who care for CF patients have recommended the use of trampolines as a physiotherapeutic tool for enhancing cardiopulmonary performance, encouraging sputum production, and improving general well-being. Despite some therapeutic and recreational benefits associated with trampoline use, papers in the general pediatric population mostly document an increased incidence of injuries, ranging from minor trauma to spinal cord injuries and even death. The aim of this review is to examine the accumulated published data regarding the use of trampolines, to assess their potential contributions and disadvantages for CF patients, and to define whether trampoline use should be recommended. An extensive search in the published medical literature retrieved approximately 60 articles that primarily dealt with trampolines, out of which only two dealt with CF. The preponderance of these articles are reports pertaining to injuries related to the use of trampolines, with only a few describing the medical, physiologic, and/or psychological benefits of trampolines. Based on the accumulated data, the presumed benefits of trampoline use for CF patients are not proven. Furthermore, the suggested benefits could be acquired using other types of exercise. Weighing the known risks of trampolines against the potential benefits that are not unique to this modality suggests that the use of trampolines for CF should not be recommended.

Activities and sources of beta-lactamase in sputum from patients with bronchiectasis.

PubMed Central

Dragicevic, P; Hill, S L; Burnett, D; Merrikin, D; Stockley, R A

1989-01-01

beta-Lactamase activity was measured in secretions from patients with bronchiectasis. Of 28 sputum samples, 23 contained measurable amounts of activity; values were significantly higher (P less than 0.01) in purulent samples than in mucoid or mucopurulent samples. beta-Lactamase activity was usually present in saliva collected before and between sputum expectorations, although values for sputum were higher than for either group of saliva samples (P less than 0.025 and P less than 0.005, respectively). This difference suggests that at least part of sputum beta-lactamase activity originates in the bronchial tree. Detailed microbiological study of a further eight specimens (seven were beta-lactamase positive) led to the isolation of Haemophilus influenzae from six, although only two of these isolates were beta-lactamase positive. Several other beta-lactamase-producing organisms were also isolated, including Staphylococcus aureus (n = 3), Escherichia coli (n = 1), Proteus spp. (n = 1), and Bacteroides spp. (n = 3). Size-exclusion high-performance liquid chromatography of the sputum showed several peaks of beta-lactamase activity which usually coeluted in fractions similar to those of their beta-lactamase-positive isolates. Therefore, sources of sputum beta-lactamases are often bacteria not considered truly pathogenic or not isolated during routine bacteriological assessment. These observations should be considered when embarking on antimicrobial therapy in bronchiectatic patients and suggest that increased dosages of penicillins are indicated. PMID:2663911

Phase I Studies of Acebilustat: Pharmacokinetics, Pharmacodynamics, Food Effect, and CYP3A Induction.

PubMed

Elborn, J S; Bhatt, L; Grosswald, R; Ahuja, S; Springman, E B

2017-01-01

Acebilustat is a new once-daily oral antiinflammatory drug in development for treatment of cystic fibrosis (CF) and other diseases. It is an inhibitor of leukotriene A4 hydrolase; therefore, production of leukotriene B4 (LTB4) in biological fluids provides a direct measure of the pharmacodynamic (PD) response to acebilustat treatment. Here we compare the pharmacokinetics (PK) and PD between CF patients and healthy volunteers, and investigate the food effect and CYP3A4 induction in healthy volunteers. No significant differences between study populations were observed for peak plasma level (C max ) or exposure (AUC). In healthy volunteers, a shift in time to C max (T max ) was observed after a high-fat meal, but there was no change in AUC. LTB4 production was reduced in the blood of both populations and in sputum from CF patients. Acebilustat did not induce CYP3A4. These results support continued clinical study of once-daily oral acebilustat in CF at doses of 50 and 100 mg. © 2016 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Biomarkers for Cystic Fibrosis Drug Development

PubMed Central

Muhlebach, Marianne S.; Clancy, JP; Heltshe, Sonya L.; Ziady, Assem; Kelley, Tom; Accurso, Frank; Pilewski, Joseph; Mayer-Hamblett, Nicole; Joseloff, Elizabeth; Sagel, Scott D.

2016-01-01

Purpose To provide a review of the status of biomarkers in cystic fibrosis drug development, including regulatory definitions and considerations, a summary of biomarkers in current use with supportive data, current gaps, and future needs. Methods Biomarkers are considered across several areas of CF drug development, including cystic fibrosis transmembrane conductance regulator modulation, infection, and inflammation. Results Sweat chloride, nasal potential difference, and intestinal current measurements have been standardized and examined in the context of multicenter trials to quantify CFTR function. Detection and quantification of pathogenic bacteria in CF respiratory cultures (e.g.: Pseudomonas aeruginosa) is commonly used in early phase antimicrobial clinical trials, and to monitor safety of therapeutic interventions. Sputum (e.g.: neutrophil elastase, myeloperoxidase, calprotectin) and blood biomarkers (e.g.: C reactive protein, calprotectin, serum amyloid A) have had variable success in detecting response to inflammatory treatments. Conclusions Biomarkers are used throughout the drug development process in CF, and many have been used in early phase clinical trials to provide proof of concept, detect drug bioactivity, and inform dosing for later-phase studies. Advances in the precision of current biomarkers, and the identification of new biomarkers with ‘omics-based technologies, are needed to accelerate CF drug development. PMID:28215711

The clinical presentations of pulmonary aspergillosis in children with cystic fibrosis - preliminary report.

PubMed

Walicka-Serzysko, Katarzyna; Sands, Dorota

2015-01-01

Pulmonary aspergillosis is a very serious complication in cystic fibrosis (CF) patients due to the great variety of its clinical presentations and the fact that it worsens the prognosis. We can distinguish the following: Aspergillus colonization (AC), Aspergillus infection (AI) and allergic bronchopulmonary aspergillosis (ABPA). Aspergillus colonization (AC) is defined as isolation of Aspergillus spp. from 50% ormore sputum samples over six months to one year without observing deterioration in lung function and an increase in such respiratory symptoms as cough. Aspergillus infection (AI) is diagnosed in subjects with Aspergillus colonization and a decline in lung function, respiratory exacerbation with and without cough or with an incomplete response to a 2-4 week course of appropriate broad-spectrum antibiotics. Aspergillus can also cause allergic bronchopulmonary aspergillosis (ABPA). The classic diagnostic criteria of allergic bronchopulmonary aspergillosis in cystic fibrosis have been established during the Cystic Fibrosis Foundation Conference in 2001. To establish the prevalence of pulmonary aspergillosis in children with cystic fibrosis under the care of our centre and to investigate the potential predisposing factors to Aspergillus infection (AI) and allergic bronchopulmonary aspergillosis (ABPA). An analysis was conducted of the medical documentation of 374 children aged 0-18 years monitored regularly in the Cystic Fibrosis Centre of the Institute of Mother and Child in Warsaw from 01.01.2010 to 31.08.2014. We selected 13 patients who presented an evidently worsening clinical status and course of the bronchopulmonary disease (decline in lung function parameters, respiratory exacerbations with increased cough, new or recent abnormalities in chest imaging) despite standard treatment with a high calorie diet, supplementation of pancreatic enzymes and vitamins, dornase alpha, inhaled and/or oral antibiotics, inhaled or oral corticosteroids, bronchodilators, physiotherapy. In this group of 13 CF children Aspergillus fumigatus was isolated from sputum. They represented 3.5% of the patients treated in our centre. Pulmonary aspergillosis was analyzed in relation to the age, sex, genotype, exocrine pancreatic insufficiency, body mass index, pulmonary function, microbiological examination of sputum, pulmonary complications and therapies. The mean age was 10.7 years (range 4.5-16.3). Only one child was under the age of six years. Patients were divided into 3 groups: patients with Aspergillus infection (AI), patients with allergic bronchopulmonary aspergillosis (ABPA), and a patient with Aspergillus infection and bronchopulmonary aspergillosis. Aspergillus infection (AI) was diagnosed in 9 cases (2.4%) and allergic bronchopulmonary aspergillosis (ABPA) in 3 (0.8%). One patient was treated with corticosteroids, because of allergic bronchopulmonary aspergillosis (ABPA) and after 8 months he developed Aspergillus infection (AI).n Most of the children were homo- or heterozygous for mutation F508del. Pancreatic insufficiency was recognized in all the children with ABPA, most of those with AI (8/9) and in one boy with ABPA and AI. Most of the patients had chronic respiratory colonization of Staphylococcus aureus and Pseudomonas aeruginosa. Children with AI were older (mean age:12.4), had a worse nutritional status (three of them had aBMI 3rd percentile), poorer lung function (five had severe lung disease *FEV1 40%*, complications occurred in one of the underlying diseases *haemoptysis, CFRD - Cystic Fibrosis Related Diabetes*, two of them had vascuport inserted due to the need for frequent intravenous antibiotic therapy. All the patients received inhaled antibiotics. A long-term oral azithromycin regime was applied in all the children with allergic bronchopulmonary aspergillosis, in most of those with Aspergillus infection *6,9* and in one boy with ABPA and AI. In three patients diagnosed with Aspergillus infection, antifungal treatment did not give any clinical or radiological improvement. They underwent surgical resection in the Department of Thoracic Surgery in Rabka (Poland). One patient had pneumonectomy and two underwent lobectomies. One boy had lung transplantation in Rigshospitalet in Copenhagen nine months after being diagnosed with Aspergillus infection. Since pulmonary aspergillosis is a very serious complication in CF children, it seems reasonable to include screening for early detection of Aspergillus colonization in the annual assessment of CF patients who are over 6 years old. Due to the small sample size and retrospective design of our analysis, the identification of risk factors of pulmonary aspergillosis in CF children require further prospective studies. .

Comparative performance of Aspergillus galactomannan ELISA and PCR in sputum from patients with ABPA and CPA.

PubMed

Fayemiwo, Samuel; Moore, Caroline B; Foden, Philip; Denning, David W; Richardson, Malcolm D

2017-09-01

Galactomannan (GM) and Aspergillus DNA detection are useful tools for the diagnosis of invasive pulmonary aspergillosis (IPA), primarily in blood and bronchoscopy samples. This study aimed to evaluate the utility of both markers for detection of Aspergillus in sputum from patients with allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis (CPA). ABPA or CPA demographic patient data were retrieved. This retrospective observational audit included 159 patients with at least one sputum pair. 223 sputum sample pairs were analysed, as well as six control samples for GM only. Real time PCR was performed following sputum DNA extraction using the MycAssay™ Aspergillus kit and cycle thresholds were subtracted from 38 to give positive values (transformed Ct, TCt). The mean age of the patients was 61.81years (SD: ±11.06; range 29-100). One hundred and twenty-six (79.2%) had CPA. Cultures were positive for fungi in 13.1% of the samples, and A. fumigatus was the commonest (11.9%) fungus isolated. Receiver operating characteristic (ROC curve) analysis of sputum GM comparing TCt of >0.0, and >2.0 to derive GMI cut-off values showed a cut-off of 6.5. About 50% of sputa with strongly positive PCR values had GM values>6.5. Two of six (33%) control samples had GM indices>6.5. It is not clear that GM determinations in sputum are useful for diagnosis of either CPA or ABPA, or following therapy. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Evaluation of genotype MTBDRplus VER 2.0 line probe assay for the detection of MDR-TB in smear positive and negative sputum samples.

PubMed

Meaza, Abyot; Kebede, Abebaw; Yaregal, Zelalem; Dagne, Zekarias; Moga, Shewki; Yenew, Bazezew; Diriba, Getu; Molalign, Helina; Tadesse, Mengistu; Adisse, Desalegn; Getahun, Muluwork; Desta, Kassu

2017-04-17

Multi drug resistant tuberculosis (MDR-TB) poses formidable challenges to TB control due to its complex diagnostic and treatment challenges and often associated with a high rate of mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Line Probe Assay (LPA) is a qualitative in vitro diagnostic test based on DNA-STRIP technology for the identification of the M. tuberculosis complex and its resistance to rifampicin (RMP) and/or isoniazid (INH). Hain Lifescience, GmbH, Germany has improved the sensitivity of Genotype MTBDRplus VER 2.0 LPA for the detection of MDR-TB; with the possibility of applying the tool in smear negative sputum samples. A cross sectional study was conducted on 274 presumptive MDR-TB patients referred to the National TB Reference Laboratory (NTRL), Ethiopian Public Health Institute (EPHI) who submitted sputum samples for laboratory diagnosis of drug resistant-TB testing. Seventy-two smear and culture positive samples processed in smear positive direct LPA category and 197 smear negative sputum samples were processed for direct LPA. Among the smear negative samples 145 (73.6%) were culture negative and 26 (13.2%) were culture positive. All specimens were processed using NALC-NaOH method and ZN smear microscopy done from sediments. Genotype MTBDRplus VER 2.0 done from processed sputum sediments and the result was compared against the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay was determined and P-value <0.05 was considered as statistically significant. The sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 LPA were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. The sensitivity, specificity, PPV and NPV of Genotype MTBDR plus VER 2.0 LPA were 77.8, 97.2, 82.4 and 97.2%, respectively, for the detection of M. tuberculosis from direct smear negative sputum samples. Fourteen (53.8%) samples had valid results with LPA among the 26 smear negative culture positive samples. The remaining 8 (30.8%) and 4 (15.4%) were invalid and negative with LPA, respectively. The sensitivity and specificity of Genotype MTBDRplus VER 2.0 LPA were 100% for the detection of MDR-TB among 14 direct smear negative and culture positive sputum samples. The most common mutations associated with RMP and INH resistance were S531L and S315TL, respectively. A single rare mutation (C15T/A16G) was detected for INH resistance. The diagnostic performance of Genotype MTBDRplus VER 2.0 LPA in direct smear positive sputum sample was highly sensitive and specific for early detection of MDR-TB. However, the diagnostic performance of this molecular assay in direct smear negative sputum sample was low and showed a high level of invalid results for detection of M. tuberculosis and its resistance to RMP and/or INH so it is unlikely to implement Genotype MTBDRplus VER 2.0 for the detection of MDR-TB in direct smear negative sample in our routine settings. The sensitivity of the assay should be improved for detection of MDR-TB in direct smear negative sputum specimens.

Lung structure and function similarities between primary ciliary dyskinesia and mild cystic fibrosis: a pilot study.

PubMed

Maglione, Marco; Montella, Silvia; Mollica, Carmine; Carnovale, Vincenzo; Iacotucci, Paola; De Gregorio, Fabiola; Tosco, Antonella; Cervasio, Mariarosaria; Raia, Valeria; Santamaria, Francesca

2017-04-12

Primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) are increasingly compared. There are no chest magnetic resonance imaging (MRI) comparative studies of PCD and CF. We assessed clinical, functional, microbiological and MRI findings in PCD and mild CF patients in order to evaluate different expression of lung disease. Twenty PCD (15.1 years) and 20 CF subjects with mild respiratory impairment (16 years, 70% with pancreatic insufficiency) underwent MRI, spirometry, and sputum cultures when clinically stable. MRI was scored using the modified Helbich system. PCD was diagnosed later than CF (9.9 versus 0.6 years, p = 0.03), despite earlier symptoms (0.1 versus 0.6 years, p = 0.02). In the year preceding the study, patients from both groups underwent two systemic antibiotic courses (p = 0.48). MRI total scores were 11.6 ± 0.7 and 9.1 ± 1 in PCD and CF, respectively. FEV 1 and FVC Z-scores were -1.75 (range, -4.6-0.7) and -0.6 (-3.9-1.8) in PCD, and -0.9 (range, -5.4-2.3) and -0.3 (-3.4-2.5) in CF, respectively. No difference was found between lung function or structure, despite a higher MRI subscore of collapse/consolidation in PCD versus CF (1.6 ± 0.1 and 0.6 ± 0.2, p < 0.001). These findings were confirmed after data-control for diagnostic delay. Pseudomonas aeruginosa and Staphylococcus aureus were more frequent in CF than in PCD (p = 0.05 and p = 0.003, respectively). MRI is a valuable radiation-free tool for comparative PCD and CF lung disease assessment. Patients with PCD may exhibit similar MRI and lung function changes as CF subjects with mild pulmonary disease. Delay in PCD diagnosis is unlikely the only determinant of similarities.

Multi-analyte profiling of inflammatory mediators in COPD sputum--the effects of processing.

PubMed

Pedersen, Frauke; Holz, Olaf; Lauer, Gereon; Quintini, Gianluca; Kiwull-Schöne, Heidrun; Kirsten, Anne-Marie; Magnussen, Helgo; Rabe, Klaus F; Goldmann, Torsten; Watz, Henrik

2015-02-01

Prior to using a new multi-analyte platform for the detection of markers in sputum it is advisable to assess whether sputum processing, especially mucus homogenization by dithiothreitol (DTT), affects the analysis. In this study we tested a novel Human Inflammation Multi Analyte Profiling® Kit (v1.0 Luminex platform; xMAP®). Induced sputum samples of 20 patients with stable COPD (mean FEV1, 59.2% pred.) were processed in parallel using standard processing (with DTT) and a more time consuming sputum dispersion method with phosphate buffered saline (PBS) only. A panel of 47 markers was analyzed in these sputum supernatants by the xMAP®. Twenty-five of 47 analytes have been detected in COPD sputum. Interestingly, 7 markers have been detected in sputum processed with DTT only, or significantly higher levels were observed following DTT treatment (VDBP, α-2-Macroglobulin, haptoglobin, α-1-antitrypsin, VCAM-1, and fibrinogen). However, standard DTT-processing resulted in lower detectable concentrations of ferritin, TIMP-1, MCP-1, MIP-1β, ICAM-1, and complement C3. The correlation between processing methods for the different markers indicates that DTT processing does not introduce a bias by affecting individual sputum samples differently. In conclusion, our data demonstrates that the Luminex-based xMAP® panel can be used for multi-analyte profiling of COPD sputum using the routinely applied method of sputum processing with DTT. However, researchers need to be aware that the absolute concentration of selected inflammatory markers can be affected by DTT. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clinical utility of capillary polymerase chain reaction for diagnosis of Cytomegalovirus pneumonia.

PubMed

Honda, J; Yonemitsu, J; Kitajima, H; Yosida, N; Fumirori, T; Oizumi, K

2001-01-01

The purpose of this retrospective study was to assess the diagnostic efficacy of CMV DNA detection by capillary PCR in patients with interstitial pneumonia. Of 882 samples taken from 363 patients, 317 were obtained from sputum, 94 from BAL fluid, 291 from blood and 180 from urine. PCR for CMV was positive in 58 samples (6.6%), with positive detection for 6.9% of sputum, 10.6% of BAL fluid, 4.1% of blood and 7.8% of urine samples. CMV pneumonia was diagnosed retrospectively in 34 (9.4%) of the 363 patients by demonstration of CMV antigen-positive cytomegalic inclusion bodies in lung tissue sections. The positive and negative predictive values were 100% (10/10) and 98.8% (83/84) for the BAL fluid samples and 95.5% (21/22) and 99.7% (294/295) for the sputum samples, respectively. Clinical sensitivity and specificity were 90.9% (10/11) and 100% (83/83) for the BAL fluid samples and 95.5% (21/22) and 99.7% (294/295) for the sputum samples, respectively. However, the blood and urine samples showed poor clinical sensitivity and low positive predictive values. We suggest that the use of capillary PCR for BAL fluid and sputum samples is very useful for diagnosing CMV pneumonia in patients with interstitial pneumonia in whom CMV pneumonia is suspected.

Pretransplant Aspergillus colonization of cystic fibrosis patients and the incidence of post-lung transplant invasive aspergillosis.

PubMed

Luong, Me-Linh; Chaparro, Cecilia; Stephenson, Anne; Rotstein, Coleman; Singer, Lianne G; Waters, Valerie; Azad, Sassan; Keshavjee, Shaf; Tullis, Elizabeth; Husain, Shahid

2014-02-15

Invasive aspergillosis (IA) is an important cause of morbidity and mortality among patients undergoing lung transplant. Cystic fibrosis-lung transplant recipients (CF-LTRs) may be at greater risk of IA following lung transplantation because of the presence of Aspergillus in their airways before transplantation. This study evaluated the impact of pretransplant Aspergillus colonization on the risk for IA among CF-LTRs. A single-center retrospective cohort study of CF-LTRs was conducted between 2006 and 2010. Respiratory tract cultures before transplantation were reviewed to identify patients with pretransplant Aspergillus colonization. Patients with positive Aspergillus sputum culture or positive bronchoalvelolar lavage (BAL) galactomannan after transplantation were classified as having colonization or disease according to the International Society of Heart and Lung Transplantation criteria. A total of 93 CF patients underwent lung transplantation. Seventy percent (65/93) of CF-LTRs had pretransplant Aspergillus colonization. Thirty-six patients had positive intraoperative Aspergillus culture from the native lung BAL. Overall, 22.5% (20/93) of CF-LTRs developed IA. Median time to IA was 42 days following transplantation. Positive intraoperative Aspergillus culture (OR 4.36, 95% CI 1.35-14.05, P=0.01) and treatment for acute cellular rejection within 90 days after transplantation (OR 3.53, 95% CI 1.03-12.15, P=0.05) were independent risk factors for IA. Antifungal prophylaxis was administered to 61% (57/93) of CF-LTRs. One-year mortality rate was 16% (15/93). IA was not associated with increased risk of death (OR 2.10, 95% CI 0.62-7.06, P=0.23). Pretransplant Aspergillus colonization is frequent among CF-LTRs and a positive intraoperative Aspergillus culture produced a fourfold higher risk of developing IA.

Guidelines for the clinical management and follow-up of infants with inconclusive cystic fibrosis diagnosis through newborn screening.

PubMed

Sermet-Gaudelus, I; Brouard, J; Audrézet, M-P; Couderc Kohen, L; Weiss, L; Wizla, N; Vrielynck, S; LLerena, K; Le Bourgeois, M; Deneuville, E; Remus, N; Nguyen-Khoa, T; Raynal, C; Roussey, M; Girodon, E

2017-12-01

Neonatal screening for cystic fibrosis (CF) can detect infants with elevated immunoreactive trypsinogen (IRT) levels and inconclusive sweat tests and/or CFTR DNA results. These cases of uncertain diagnosis are defined by (1) either the presence of at most one CF-associated cystic fibrosis transmembrane conductance regulator (CFTR) mutation with sweat chloride values between 30 and 59mmol/L or (2) two CFTR mutations with at least one of unknown pathogenic potential and a sweat chloride concentration below 60mmol/L. This encompasses various clinical situations whose progression cannot be predicted. In these cases, a sweat chloride test has to be repeated at 12 months, and if possible at 6 and 24 months of life along with extended CFTR sequencing to detect rare mutations. When the diagnosis is not definite, CFTR functional explorations may provide a better understanding of CFTR dysfunction. The initial evaluation of these infants must be conducted in dedicated CF reference centers and should include bacteriological sputum analysis, chest radiology, and fecal elastase assay. The primary care physicians in charge of these patients should be familiar with the current management of CF and should work in collaboration with CF centers. A follow-up should be performed in a CF reference center at 3, 6, and 12 months of life and every year thereafter. Any symptom indicative of CF requires immediate reevaluation of the diagnosis. These guidelines were established by the "neonatal screening and difficult diagnoses" working group of the French CF society. Their objective is to standardize the management of infants with unclear diagnosis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The Diagnostic Utility of Induced Sputum Microscopy and Culture in Childhood Pneumonia

PubMed Central

Morpeth, Susan C.; Hammitt, Laura L.; Driscoll, Amanda J.; Watson, Nora L.; Baggett, Henry C.; Brooks, W. Abdullah; Deloria Knoll, Maria; Feikin, Daniel R.; Kotloff, Karen L.; Levine, Orin S.; Madhi, Shabir A.; O’Brien, Katherine L.; Scott, J. Anthony G.; Thea, Donald M.; Adrian, Peter V.; Ahmed, Dilruba; Alam, Muntasir; Awori, Juliet O.; DeLuca, Andrea N.; Higdon, Melissa M.; Karron, Ruth A.; Kwenda, Geoffrey; Machuka, Eunice M.; Makprasert, Sirirat; McLellan, Jessica; Moore, David P.; Mwaba, John; Mwarumba, Salim; Park, Daniel E.; Prosperi, Christine; Sangwichian, Ornuma; Sissoko, Seydou; Tapia, Milagritos D.; Zeger, Scott L.; Howie, Stephen R. C.; O’Brien, Katherine L.; Levine, Orin S.; Knoll, Maria Deloria; Feikin, Daniel R.; DeLuca, Andrea N.; Driscoll, Amanda J.; Fancourt, Nicholas; Fu, Wei; Hammitt, Laura L.; Higdon, Melissa M.; Kagucia, E. Wangeci; Karron, Ruth A.; Li, Mengying; Park, Daniel E.; Prosperi, Christine; Wu, Zhenke; Zeger, Scott L.; Watson, Nora L.; Crawley, Jane; Murdoch, David R.; Brooks, W. Abdullah; Endtz, Hubert P.; Zaman, Khalequ; Goswami, Doli; Hossain, Lokman; Jahan, Yasmin; Ashraf, Hasan; Howie, Stephen R. C.; Ebruke, Bernard E.; Antonio, Martin; McLellan, Jessica; Machuka, Eunice; Shamsul, Arifin; Zaman, Syed M.A.; Mackenzie, Grant; Scott, J. Anthony G.; Awori, Juliet O.; Morpeth, Susan C.; Kamau, Alice; Kazungu, Sidi; Ominde, Micah Silaba; Kotloff, Karen L.; Tapia, Milagritos D.; Sow, Samba O.; Sylla, Mamadou; Tamboura, Boubou; Onwuchekwa, Uma; Kourouma, Nana; Toure, Aliou; Madhi, Shabir A.; Moore, David P.; Adrian, Peter V.; Baillie, Vicky L.; Kuwanda, Locadiah; Mudau, Azwifarwi; Groome, Michelle J.; Mahomed, Nasreen; Baggett, Henry C.; Thamthitiwat, Somsak; Maloney, Susan A.; Bunthi, Charatdao; Rhodes, Julia; Sawatwong, Pongpun; Akarasewi, Pasakorn; Thea, Donald M.; Mwananyanda, Lawrence; Chipeta, James; Seidenberg, Phil; Mwansa, James; wa Somwe, Somwe; Kwenda, Geoffrey; Anderson, Trevor P.; Mitchell, Joanne

2017-01-01

Abstract Background. Sputum microscopy and culture are commonly used for diagnosing the cause of pneumonia in adults but are rarely performed in children due to difficulties in obtaining specimens. Induced sputum is occasionally used to investigate lower respiratory infections in children but has not been widely used in pneumonia etiology studies. Methods. We evaluated the diagnostic utility of induced sputum microscopy and culture in patients enrolled in the Pneumonia Etiology Research for Child Health (PERCH) study, a large study of community-acquired pneumonia in children aged 1–59 months. Comparisons were made between induced sputum samples from hospitalized children with radiographically confirmed pneumonia and children categorized as nonpneumonia (due to the absence of prespecified clinical and laboratory signs and absence of infiltrate on chest radiograph). Results. One induced sputum sample was available for analysis from 3772 (89.1%) of 4232 suspected pneumonia cases enrolled in PERCH. Of these, sputum from 2608 (69.1%) met the quality criterion of <10 squamous epithelial cells per low-power field, and 1162 (44.6%) had radiographic pneumonia. Induced sputum microscopy and culture results were not associated with radiographic pneumonia, regardless of prior antibiotic use, stratification by specific bacteria, or interpretative criteria used. Conclusions. The findings of this study do not support the culture of induced sputum specimens as a diagnostic tool for pneumonia in young children as part of routine clinical practice. PMID:28575362

SIMPLE MEASURES ARE AS EFFECTIVE AS INVASIVE TECHNIQUES IN THE DIAGNOSIS OF PULMONARY TUBERCULOSIS IN MALAWI

PubMed Central

Bell, David J; Dacombe, Russell; Graham, Stephen M; Hicks, Alexander; Cohen, Danielle; Chikaonda, Tarsizio; French, Neil; Molyneux, Malcolm E; Zijlstra, Ed E; Squire, S Bertel; Gordon, Stephen B

2010-01-01

Setting Detection of smear-positive pulmonary tuberculosis (PTB) cases is vital for tuberculosis control. Methods to augment sputum collection are available but their additional benefit is uncertain in resource-limited settings. Objective To compare the diagnostic yields using five methods to obtain sputum from adults diagnosed with smear-negative PTB in Malawi. Design Self-expectorated sputum was collected under supervision for microscopy and mycobacterial culture in the study laboratory. Confirmed smear-negative patients, provided physiotherapy-assisted sputum and induced sputum followed, the next morning, by gastric washing and bronchoalveolar-lavage samples. Results 150 patients, diagnosed with smear-negative PTB by the hospital service, were screened. 39 (26%) were smear-positive from supervised self-expectorated sputum examined in the study laboratory. The remaining 111 confirmed smear-negative patients were enrolled; 89% were HIV positive. Seven additional smear-positive cases were diagnosed using the augmented sputum collection techniques. No differences were observed in the numbers of cases detected using the different methods. 44 (95.6%) of the 46 smear-positive cases could be detected from self-expectorated and physiotherapy-assisted samples Conclusions For countries like Malawi, the best use of limited resources to detect smear-positive PTB cases would be to improve the quality of self-expectorated sputum collection and microscopy. The additional diagnostic yield using bronchoalveolar-lavage after induced sputum is limited. PMID:19105886

The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing

PubMed Central

Simmons, Ann Marie; Rowneki, Mazhgan; Parmar, Heta; Cao, Yuan; Ryan, Jamie; Banada, Padmapriya P.; Deshpande, Srinidhi; Shenai, Shubhada; Gall, Alexander; Glass, Jennifer; Krieswirth, Barry; Schumacher, Samuel G.; Nabeta, Pamela; Tukvadze, Nestani; Rodrigues, Camilla; Skrahina, Alena; Tagliani, Elisa; Cirillo, Daniela M.; Davidow, Amy; Denkinger, Claudia M.; Persing, David; Kwiatkowski, Robert; Jones, Martin

2017-01-01

ABSTRACT The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. PMID:28851844

Characterization by phenotypic and genotypic methods of metallo-β-lactamase-producing Pseudomonas aeruginosa isolated from patients with cystic fibrosis.

PubMed

Li, Yongwei; Zhang, Xiaoqian; Wang, Chunxia; Hu, Yue; Niu, Xiaobin; Pei, Dongxu; He, Zhiqiang; Bi, Yongyi

2015-01-01

Pseudomonas aeruginosa continues to be a predominant cause of infections with high intrinsic resistance to antibiotics, resulting in treatment failure. P. aeruginosa is the leading cause of respiratory infections among cystic fibrosis (CF) patients. Resistance to carbapenem antibiotics among P. aeruginosa has been reported. Thus, this study was undertaken to characterize the metallo-β-lactamase (MBL) production of P. aeruginosa by phenotypic and genotypic methods. A total of 572 sputum samples were collected from cystic fibrosis patients along with the patient demographic details in a questionnaire. In total, 217 P. aeruginosa isolates were collected and an antibiogram revealed that 159 (73.3%) and 141 (64.9%) of these colonies exhibited resistance to imipenem and meropenem, respectively. Ceftazidime and tobramycin resistance were both identified in 112 (51.6%) isolates, and resistance to piperacillin-tazobactam, gatifloxacin and netilmicin was detected in 96 (44.2%) respective samples. A total of 62 (28.6%) respective samples were resistant to cefoperazone, cefepime and ceftriaxone. The least antibiotic resistance was shown to amikacin and ceftizoxime with 51 (23.5%) and 32 (14.7%) respective colonies resistant to the antibiotics. The minimum inhibitory concentration (MIC) for imipenem revealed a reduction in the MIC values. MBL screening by the zone enhancement method using ceftazidime plus EDTA discs demonstrated that 63 (56.25%) of the colonies were positive for MBL. A total of 53 (84.1%) samples expressed blaVIM and 48 (76.1%) expressed blaIMP genes, as detected by duplex polymerase chain reaction. In conclusion, carbapenem resistance is of great clinical concern in cystic fibrosis patients with P. aeruginosa infection. Therefore, mandatory regular screening and monitoring the resistance in P. aeruginosa among CF patients is required.

Evaluation of Microscopic Observation Drug Susceptibility (MODS) and the string test for rapid diagnosis of pulmonary tuberculosis in HIV/AIDS patients in Bolivia.

PubMed

Lora, Meredith H; Reimer-McAtee, Melissa J; Gilman, Robert H; Lozano, Daniel; Saravia, Ruth; Pajuelo, Monica; Bern, Caryn; Castro, Rosario; Espinoza, Magaly; Vallejo, Maya; Solano, Marco; Challapa, Roxana; Torrico, Faustino

2015-06-06

Tuberculosis (TB) is the most common opportunistic infection and the leading cause of death in HIV-positive people worldwide. Diagnosing TB is difficult, and is more challenging in resource-scarce settings where culture-based diagnostic methods rely on poorly sensitive smear microscopy by Ziehl-Neelsen stain (ZN). We performed a cross-sectional study examining the diagnostic utility of Microscopic Observation Drug Susceptibility liquid culture (MODS) versus traditional Ziehl-Neelsen staining (ZN) and Lowenstein Jensen culture (LJ) of pulmonary tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB) in HIV-infected patients in Bolivia. For sputum scarce individuals we assessed the value of the string test and induced sputum for TB diagnosis. The presence of Mycobacterium tuberculosis (Mtb) in the sputum of 107 HIV-positive patients was evaluated by ZN, LJ, and MODS. Gastric secretion samples obtained by the string test were evaluated by MODS in 102 patients. The TB-HIV co-infection rate of HIV patients with respiratory symptoms by sputum sample was 45 % (48/107); 46/48 (96 %) were positive by MODS, 38/48 (79 %) by LJ, and 30/48 (63 %) by ZN. The rate of MDRTB was 9 % (4/48). Median time to positive culture was 10 days by MODS versus 34 days by LJ (p < 0.0001). In smear-negative patients, MODS detected TB in 17/18 patients, compared to 11/18 by LJ (94.4 % vs 61.0 %, p = 0.03 %). In patients unable to produce a sputum sample without induction, the string test cultured by MODS yielded Mtb in of 9/11 (82 %) TB positive patients compared to 11/11 (100 %) with induced sputum. Of the 10 patients unable to produce a sputum sample, 4 were TB-positive by string test. MODS was faster and had a higher Mtb detection yield compared to LJ, with a greater difference in yield between the two in smear-negative patients. The string test is a valuable diagnostic technique for HIV sputum-scarce or sputum-absent patients, and should be considered as an alternative test to induced sputum to obtain sample for Mtb in resource-limited settings. Nine percent of our TB+ patients had MDRTB, which reinforces the need for rapid detection with direct drug susceptibility testing in HIV patients in Bolivia.

Acute Pain Perception During Different Sampling Methods for Respiratory Culture in Cystic Fibrosis Patients.

PubMed

Eyns, Hanneke; De Wachter, Elke; Malfroot, Anne; Vaes, Peter

2018-03-01

Reliable identification of lower respiratory tract pathogens is crucial in the management of cystic fibrosis (CF). The multitude of treatments and clinical procedures are a considerable burden and are potentially provoking pain. As part of another study (NCT02363764), investigating the bacterial yield of three sampling methods, nasal swabs (NSs), cough swabs (CSs), and (induced) sputum samples ([I]SSs), in both expectorating patients (EPs) and non-expectorating patients (NEPs) with CF, the present study aimed to explore the prevalence of respiratory culture sampling-related pain as assessed by self-report within a cohort of children and adults. Literate patients with CF (aged six years or older) completed a questionnaire on pain perception related to the three aforementioned sampling methods (No/Yes; visual analogue scale for pain [VAS-Pain] [0-10 cm]). In addition, patients were asked to rank these methods by their own preference without taking into account the presumed bacterial yield. In total, 119 questionnaires were returned. In the EPs-group, CS was most frequently (n%; mean VAS-Pain if pain [range]) reported as painful method: overall (n = 101; 12.9%; 1.8 [0.2-4.8]), children (n = 41; 22.0%; 1.4 [0.2-2.7]), and adults (n = 60; 6.7%; 2.5 [0.5-4.8]). Highest pain intensity scores were observed with NS overall (3.0%; 2.4 [0.3-6.2]) and in children (4.9%; 3.3 [0.3-6.2]), but not in adults (1.7%; 0.6 [-]).NEPs-children (n = 17) reported ISS most frequently and as most painful sampling method (17.6%; 2.0 [1.0-4.0]). The only NEP-adult did not perceive pain. NEPs preferred NS > CS > ISS (61.1%, 33.3%, 5.6%, respectively [P = 0.001]) as primary sampling method, whereas EPs preferred SS > NS > CS (65.7%, 26.3%, 8.1%, respectively [P < 0.0001]). Patients' preference for a specific method inversely correlated to pain perception and intensity in EPs (φ = -0.155 [P = 0.007] and ρ = -0.926 [P = 0.008], respectively), but not in NEPs (φ = -0.226 [P = 0.097] and ρ = -0.135 [P = 0.798], respectively). A relatively large range of pain experiences was observed in patients with CF during respiratory culture sampling, which underlines the importance of individual pain assessment. Nevertheless, clinicians can confidently choose the sampling method based on validity over patients' preference. Copyright © 2017 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.

Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-01

Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1β) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

Differences in fungi present in induced sputum samples from asthma patients and non-atopic controls: a community based case control study

PubMed Central

2013-01-01

Background There is emerging evidence for the presence of an extensive microbiota in human lungs. It is not known whether variations in the prevalence of species of microbiota in the lungs may have aetiological significance in respiratory conditions such as asthma. The aim of the study was to undertake semi-quantitative analysis of the differences in fungal species in pooled sputum samples from asthma patients and controls. Methods Induced sputum samples were collected in a case control study of asthma patients and control subjects drawn from the community in Wandsworth, London. Samples from both groups were pooled and then tested for eukaryotes. DNA was amplified using standard PCR techniques, followed by pyrosequencing and comparison of reads to databases of known sequences to determine in a semi-quantitative way the percentage of DNA from known species in each of the two pooled samples. Results A total of 136 fungal species were identified in the induced sputum samples, with 90 species more common in asthma patients and 46 species more common in control subjects. Psathyrella candolleana, Malassezia pachydermatis, Termitomyces clypeatus and Grifola sordulenta showed a higher percentage of reads in the sputum of asthma patients and Eremothecium sinecaudum, Systenostrema alba, Cladosporium cladosporioides and Vanderwaltozyma polyspora showed a higher percentage of reads in the sputum of control subjects. A statistically significant difference in the pattern of fungi that were present in the respective samples was demonstrated using the Phylogenetic (P) test (P < 0.0001). Conclusion This study is novel in providing evidence for the widespread nature of fungi in the sputum of healthy and asthmatic individuals. Differences in the pattern of fungi present in asthma patients and controls merit further investigation. Of particular interest was the presence of Malassezia pachydermatis, which is known to be associated with atopic dermatitis. PMID:23384395

Differences in fungi present in induced sputum samples from asthma patients and non-atopic controls: a community based case control study.

PubMed

van Woerden, Hugo Cornelis; Gregory, Clive; Brown, Richard; Marchesi, Julian Roberto; Hoogendoorn, Bastiaan; Matthews, Ian Price

2013-02-05

There is emerging evidence for the presence of an extensive microbiota in human lungs. It is not known whether variations in the prevalence of species of microbiota in the lungs may have aetiological significance in respiratory conditions such as asthma. The aim of the study was to undertake semi-quantitative analysis of the differences in fungal species in pooled sputum samples from asthma patients and controls. Induced sputum samples were collected in a case control study of asthma patients and control subjects drawn from the community in Wandsworth, London. Samples from both groups were pooled and then tested for eukaryotes. DNA was amplified using standard PCR techniques, followed by pyrosequencing and comparison of reads to databases of known sequences to determine in a semi-quantitative way the percentage of DNA from known species in each of the two pooled samples. A total of 136 fungal species were identified in the induced sputum samples, with 90 species more common in asthma patients and 46 species more common in control subjects. Psathyrella candolleana, Malassezia pachydermatis, Termitomyces clypeatus and Grifola sordulenta showed a higher percentage of reads in the sputum of asthma patients and Eremothecium sinecaudum, Systenostrema alba, Cladosporium cladosporioides and Vanderwaltozyma polyspora showed a higher percentage of reads in the sputum of control subjects. A statistically significant difference in the pattern of fungi that were present in the respective samples was demonstrated using the Phylogenetic (P) test (P < 0.0001). This study is novel in providing evidence for the widespread nature of fungi in the sputum of healthy and asthmatic individuals. Differences in the pattern of fungi present in asthma patients and controls merit further investigation. Of particular interest was the presence of Malassezia pachydermatis, which is known to be associated with atopic dermatitis.

Linear rheology as a potential monitoring tool for sputum in patients with Chronic Obstructive Pulmonary Disease (COPD).

PubMed

Nettle, C J; Jenkins, L; Curtis, D; Badiei, N; Lewis, K; Williams, P R; Daniels, D R

2018-01-01

The rheological properties of sputum may influence lung function and become modified in disease. This study aimed to correlate the viscoelastic properties of sputum with clinical data on the severity of disease in patients with chronic obstructive pulmonary disease (COPD). Sputum samples from COPD patients were investigated using rheology, simple mathematical modelling and Scanning Electron Microscopy (SEM). The samples were all collected from patients within two days of their admission to Prince Philip Hospital due to an exacerbation of their COPD. Oscillatory and creep rheological techniques were used to measure changes in viscoelastic properties at different frequencies over time. COPD sputum was observed to behave as a viscoelastic solid at all frequencies studied. Comparing the rheology of exacerbated COPD sputum with healthy sputum (not diagnosed with a respiratory disease) revealed significant differences in response to oscillatory shear and creep-recovery experiments, which highlights the potential clinical benefits of better understanding sputum viscoelasticity. A common power law model G(t)=G0(tτ0)-m was successfully fitted to experimental rheology data over the range of frequencies studied. A comparison between clinical data and the power law index m obtained from rheology, suggested that an important possible future application of this parameter is as a potential biomarker for COPD severity.

IRON-BINDING AND STORAGE PROTEINS IN SPUTUM

EPA Science Inventory

Induced sputum (IS) and bronchoalveolar lavage (BAL) sample different lung compartments, with IS obtaining secretions from the surfaces of the bronchial airways and BAL sampling secretions from the alveolar airspaces. Deposition of iron-containing particulate matter occurs prefer...

Development of Antigen Detection Assay for Diagnosis of Tuberculosis Using Sputum Samples

PubMed Central

Pereira Arias-Bouda, Lenka M.; Nguyen, Lan N.; Ho, Ly M.; Kuijper, Sjoukje; Jansen, Henk M.; Kolk, Arend H. J.

2000-01-01

The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 104 bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 104 M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 μg of LAM/ml. The detection limit in sputum pretreated with N-acetyl-l-cysteine and proteinase K was 104 M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis. PMID:10834989

Effect of freezing of sputum samples on flow cytometric analysis of lymphocyte subsets.

PubMed

Jaksztat, E; Holz, O; Paasch, K; Kelly, M M; Hargreave, F E; Cox, G; Magnussen, H; Jörres, R A

2004-08-01

Sputum samples should be processed shortly after induction to prevent cell degradation. For intermediate storage, freezing of homogenised samples or immediate fixation have been shown to be suitable for cytospins. The aim of this study was to investigate whether freezing or immediate fixation of sputum affect the analysis of lymphocyte subsets by flow cytometry. Selected plugs from 24 sputum samples were homogenised. One aliquot was processed immediately and analysed by flow cytometry. A second aliquot was homogenised, frozen at -20 C after addition of dimethylsulfoxide and stored for a median time of 6 days. In six samples a third aliquot was fixed in formalin after induction and stored for up to 72 h before further processing. Compared to immediate processing, percentages of total lymphocytes and T-suppressor cells were elevated after being frozen, with a minor decrease in the T4/T8 ratio. Proportions of total lymphocytes, T-helper and T-suppressor cells correlated between native and frozen samples, intra-class correlation coefficients being 0.74, 0.85 and 0.70, respectively. The formalin-fixed aliquots could not be analysed with the antibodies used. In conclusion, freezing seems to be a suitable technique to store sputum samples for flow cytometry of CD3, CD4 and CD8 lymphocyte subsets. Its effects were minor compared to the variation between subjects.

Anti-inflammatory effects of salmeterol/fluticasone propionate 50/250 mcg combination therapy in Japanese patients with chronic obstructive pulmonary disease

PubMed Central

Asai, Kazuhisa; Kobayashi, Akihiro; Makihara, Yukio; Johnson, Malcolm

2015-01-01

Purpose Using sputum neutrophils as the primary measure, and other inflammation biomarkers, this study evaluated the anti-inflammatory effects of the combination salmeterol 50 mcg and fluticasone propionate 250 mcg (SFC 250) in Japanese patients with chronic obstructive pulmonary disease (COPD). Patients and methods Patients were treated in a randomized, double-blind, parallel group, placebo-controlled trial with SFC 250 twice daily (n=26) or placebo (n=26) for 12 weeks. At the start and end of treatment, inflammation biomarkers (sputum and serum), lung function, and health status (COPD Assessment Test [CAT] questionnaire) were measured. Results Although a numerical decrease in differential neutrophil count was observed from baseline, SFC 250 did not significantly reduce sputum neutrophils compared with placebo, nor were there significant changes from baseline in the other biomarkers (sputum or serum), lung function, or CAT, versus placebo. Squamous epithelial cell contamination in some sputum samples rendered them unacceptable for analysis, which reduced the sample size to n=19 (SFC 250) and n=10 (placebo). However, inclusion of contaminated samples did not affect the overall trend of the outcome. Ad hoc bootstrap statistical analysis showed a 27.9% (SFC 250) and 1.3% (placebo) decrease in sputum neutrophils. Sputum IL-8 decreased by 43.2% after SFC 250 but increased by 48.3% with placebo. Responder analyses showed 42% of patients had ≥20% decrease in neutrophils from baseline; and 47% of patients had a ≥200 pg/mL change in sputum IL-8 following SFC 250 versus 20% after placebo; both changes are considered clinically relevant. Conclusion This study provides additional information about inflammation in Japanese COPD patients and is the first to study the anti-inflammatory effects of SFC 250 in this context and population. In the primary analysis, SFC 250 did not produce significant changes from baseline in sputum neutrophil levels or other sputum or serum inflammatory markers compared with placebo. Secondary ad hoc statistical analysis showed that SFC 250 reduced the number of sputum neutrophils and IL-8 compared with placebo. PMID:25945045

Anti-inflammatory effects of salmeterol/fluticasone propionate 50/250 mcg combination therapy in Japanese patients with chronic obstructive pulmonary disease.

PubMed

Asai, Kazuhisa; Kobayashi, Akihiro; Makihara, Yukio; Johnson, Malcolm

2015-01-01

Using sputum neutrophils as the primary measure, and other inflammation biomarkers, this study evaluated the anti-inflammatory effects of the combination salmeterol 50 mcg and fluticasone propionate 250 mcg (SFC 250) in Japanese patients with chronic obstructive pulmonary disease (COPD). Patients were treated in a randomized, double-blind, parallel group, placebo-controlled trial with SFC 250 twice daily (n=26) or placebo (n=26) for 12 weeks. At the start and end of treatment, inflammation biomarkers (sputum and serum), lung function, and health status (COPD Assessment Test [CAT] questionnaire) were measured. Although a numerical decrease in differential neutrophil count was observed from baseline, SFC 250 did not significantly reduce sputum neutrophils compared with placebo, nor were there significant changes from baseline in the other biomarkers (sputum or serum), lung function, or CAT, versus placebo. Squamous epithelial cell contamination in some sputum samples rendered them unacceptable for analysis, which reduced the sample size to n=19 (SFC 250) and n=10 (placebo). However, inclusion of contaminated samples did not affect the overall trend of the outcome. Ad hoc bootstrap statistical analysis showed a 27.9% (SFC 250) and 1.3% (placebo) decrease in sputum neutrophils. Sputum IL-8 decreased by 43.2% after SFC 250 but increased by 48.3% with placebo. Responder analyses showed 42% of patients had ≥20% decrease in neutrophils from baseline; and 47% of patients had a ≥200 pg/mL change in sputum IL-8 following SFC 250 versus 20% after placebo; both changes are considered clinically relevant. This study provides additional information about inflammation in Japanese COPD patients and is the first to study the anti-inflammatory effects of SFC 250 in this context and population. In the primary analysis, SFC 250 did not produce significant changes from baseline in sputum neutrophil levels or other sputum or serum inflammatory markers compared with placebo. Secondary ad hoc statistical analysis showed that SFC 250 reduced the number of sputum neutrophils and IL-8 compared with placebo.

Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum.

PubMed

Kopf, Sebastian H; Sessions, Alex L; Cowley, Elise S; Reyes, Carmen; Van Sambeek, Lindsey; Hu, Yang; Orphan, Victoria J; Kato, Roberta; Newman, Dianne K

2016-01-12

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.

Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum

NASA Astrophysics Data System (ADS)

Kopf, Sebastian H.; Sessions, Alex L.; Cowley, Elise S.; Reyes, Carmen; Van Sambeek, Lindsey; Hu, Yang; Orphan, Victoria J.; Kato, Roberta; Newman, Dianne K.

2016-01-01

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.

The Diagnostic Utility of Induced Sputum Microscopy and Culture in Childhood Pneumonia.

PubMed

Murdoch, David R; Morpeth, Susan C; Hammitt, Laura L; Driscoll, Amanda J; Watson, Nora L; Baggett, Henry C; Brooks, W Abdullah; Deloria Knoll, Maria; Feikin, Daniel R; Kotloff, Karen L; Levine, Orin S; Madhi, Shabir A; O'Brien, Katherine L; Scott, J Anthony G; Thea, Donald M; Adrian, Peter V; Ahmed, Dilruba; Alam, Muntasir; Awori, Juliet O; DeLuca, Andrea N; Higdon, Melissa M; Karron, Ruth A; Kwenda, Geoffrey; Machuka, Eunice M; Makprasert, Sirirat; McLellan, Jessica; Moore, David P; Mwaba, John; Mwarumba, Salim; Park, Daniel E; Prosperi, Christine; Sangwichian, Ornuma; Sissoko, Seydou; Tapia, Milagritos D; Zeger, Scott L; Howie, Stephen R C

2017-06-15

Sputum microscopy and culture are commonly used for diagnosing the cause of pneumonia in adults but are rarely performed in children due to difficulties in obtaining specimens. Induced sputum is occasionally used to investigate lower respiratory infections in children but has not been widely used in pneumonia etiology studies. We evaluated the diagnostic utility of induced sputum microscopy and culture in patients enrolled in the Pneumonia Etiology Research for Child Health (PERCH) study, a large study of community-acquired pneumonia in children aged 1-59 months. Comparisons were made between induced sputum samples from hospitalized children with radiographically confirmed pneumonia and children categorized as nonpneumonia (due to the absence of prespecified clinical and laboratory signs and absence of infiltrate on chest radiograph). One induced sputum sample was available for analysis from 3772 (89.1%) of 4232 suspected pneumonia cases enrolled in PERCH. Of these, sputum from 2608 (69.1%) met the quality criterion of <10 squamous epithelial cells per low-power field, and 1162 (44.6%) had radiographic pneumonia. Induced sputum microscopy and culture results were not associated with radiographic pneumonia, regardless of prior antibiotic use, stratification by specific bacteria, or interpretative criteria used. The findings of this study do not support the culture of induced sputum specimens as a diagnostic tool for pneumonia in young children as part of routine clinical practice. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Lot quality assurance sampling of sputum acid-fast bacillus smears for assessing sputum smear microscopy centers.

PubMed

Selvakumar, N; Murthy, B N; Prabhakaran, E; Sivagamasundari, S; Vasanthan, Samuel; Perumal, M; Govindaraju, R; Chauhan, L S; Wares, Fraser; Santha, T; Narayanan, P R

2005-02-01

Assessment of 12 microscopy centers in a tuberculosis unit by blinded checking of eight sputum smears selected by using a lot quality assurance sampling (LQAS) method and by unblinded checking of all positive and five negative slides, among the slides examined in a month in a microscopy centre, revealed that the LQAS method can be implemented in the field to monitor the performance of acid-fast bacillus microscopy centers in national tuberculosis control programs.

Lot Quality Assurance Sampling of Sputum Acid-Fast Bacillus Smears for Assessing Sputum Smear Microscopy Centers

PubMed Central

Selvakumar, N.; Murthy, B. N.; Prabhakaran, E.; Sivagamasundari, S.; Vasanthan, Samuel; Perumal, M.; Govindaraju, R.; Chauhan, L. S.; Wares, Fraser; Santha, T.; Narayanan, P. R.

2005-01-01

Assessment of 12 microscopy centers in a tuberculosis unit by blinded checking of eight sputum smears selected by using a lot quality assurance sampling (LQAS) method and by unblinded checking of all positive and five negative slides, among the slides examined in a month in a microscopy centre, revealed that the LQAS method can be implemented in the field to monitor the performance of acid-fast bacillus microscopy centers in national tuberculosis control programs. PMID:15695704

Antibiotic exposure and interpersonal variance mask the effect of ivacaftor on respiratory microbiota composition.

PubMed

Peleg, Anton Y; Choo, Jocelyn M; Langan, Katherine M; Edgeworth, Deirdre; Keating, Dominic; Wilson, John; Rogers, Geraint B; Kotsimbos, Tom

2018-01-01

G551D is a class III mutation of the cystic fibrosis transmembrane regulator (CFTR) that results in impaired chloride channel function in cystic fibrosis (CF). Ivacaftor, a CFTR-potentiating agent improves sweat chloride, weight, lung function, and pulmonary exacerbation rate in CF patients with G551D mutations, but its effect on the airway microbiome remains poorly characterised. Twenty CF patients with at least one G551D mutation from a single centre were recruited to a 4month double-blind, placebo-controlled, crossover study of ivacaftor with 28days of active treatment. Sputum microbiota composition was assessed by 16S rRNA gene amplicon sequencing and quantitative PCR at five key time points, along with regular clinical review, respiratory function assessment, and peripheral blood testing. No significant difference in microbiota composition was observed in subjects following ivacaftor treatment or placebo (PERMANOVA P=0.95, square root ECV=-4.94, 9479 permutations). Microbiota composition variance was significantly greater between subjects, than within subjects over time (P<0.0001, Mann Whitney U test), and an additional within-patient paired assessment of microbiota similarity was therefore performed. Again, change in microbiota composition was not significantly greater during treatment with ivacaftor compared to placebo (Wilcoxon test, P=0.51). A significant change in microbiota composition was however associated with any change in antibiotic exposure, regardless of whether ivacaftor or placebo was administered (P=0.006). In a small, subgroup analysis of subjects whose antibiotic exposure did not change within the study period, a significant reduction in total bacterial load was observed during treatment with ivacaftor (P=0.004, two-tailed paired Student's t-test). The short-term impact of ivacaftor therapy on sputum microbiota composition in patients with G551D mutations are modest compared to those resulting from antibiotic exposure, and may be masked by changes in antibiotic treatment regimen. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Xpert MTB/RIF diagnosis of childhood tuberculosis from sputum and stool samples in a high TB-HIV-prevalent setting.

PubMed

Orikiriza, Patrick; Nansumba, Margaret; Nyehangane, Dan; Bastard, Mathieu; Mugisha, Ivan Taremwa; Nansera, Denis; Mwanga-Amumpaire, Juliet; Boum, Yap; Kumbakumba, Elias; Bonnet, Maryline

2018-05-08

The Xpert MTB/RIF assay is a major advance for diagnosis of tuberculosis (TB) in high-burden countries but is limited in children by their difficulty to produce sputum. We investigated TB in sputum and stool from children with the aim of improving paediatric TB diagnosis. A prospective cohort of children with presumptive TB, provided two sputum or induced sputum at enrolment in a regional referral hospital in Uganda. Stool was collected from those started on TB treatment. All specimen were tested for Xpert MTB/RIF, mycobacteria growth indicator tube (MGIT), Lowenstein Jensen cultures and microscopy (except stool). We compared TB detection between age categories and assessed the performance of Xpert MTB/RIF in sputum and stool. Of the 392 children enrolled, 357 (91.1%) produced at least one sputum sample. Sputum culture yield was 13/357 (3.6%): 3/109 (2.6%), 3/89 (3.2%), 3/101 (2.6%) and 4/44 (8.2%) among children of < 2, 2-5, ≥ 5-10 and > 10 years, respectively (p = 0.599). Xpert MTB/RIF yield was 14/350 (4.0%): 3/104 (2.9%), 4/92 (4.3%), 3/88 (2.9%) and 4/50 (.0%), respectively (p = 0.283). Sensitivity and specificity of Xpert MTB/RIF in sputum against sputum culture were 90.9% (95% CI 58.7-99.8) and 99.1% (99.1-99.8). In stool, it was 55.6% (21.2-86.3) and 98.2% (98.2-100) against Xpert MTB/RIF and culture in sputum. Only a minority of children had microbiologically confirmed TB with a higher proportion in children above 10 years. Although sensitivity of Xpert MTB/RIF in stool was low, with good optimization, it might be a good alternative to sputum in children.

Pharmacokinetics and Pharmacodynamics of Aerosolized Antibacterial Agents in Chronically Infected Cystic Fibrosis Patients

PubMed Central

2014-01-01

SUMMARY Bacteria adapt to growth in lungs of patients with cystic fibrosis (CF) by selection of heterogeneously resistant variants that are not detected by conventional susceptibility testing but are selected for rapidly during antibacterial treatment. Therefore, total bacterial counts and antibiotic susceptibilities are misleading indicators of infection and are not helpful as guides for therapy decisions or efficacy endpoints. High drug concentrations delivered by aerosol may maximize efficacy, as decreased drug susceptibilities of the pathogens are compensated for by high target site concentrations. However, reductions of the bacterial load in sputum and improvements in lung function were within the same ranges following aerosolized and conventional therapies. Furthermore, the use of conventional pharmacokinetic/pharmacodynamic (PK/PD) surrogates correlating pharmacokinetics in serum with clinical cure and presumed or proven eradication of the pathogen as a basis for PK/PD investigations in CF patients is irrelevant, as minimization of systemic exposure is one of the main objectives of aerosolized therapy; in addition, bacterial pathogens cannot be eradicated, and chronic infection cannot be cured. Consequently, conventional PK/PD surrogates are not applicable to CF patients. It is nonetheless obvious that systemic exposure of patients, with all its sequelae, is minimized and that the burden of oral treatment for CF patients suffering from chronic infections is reduced. PMID:25278574

Clinical and immunological characteristics associated with the presence of protozoa in sputum smears.

PubMed

Martínez-Girón, Rafael; van Woerden, Hugo Cornelis

2013-01-01

The objective of this study is to assess the relationship between protozoa in spontaneously expectorated sputum samples and a range of clinical and immunological variables. Clinical details including age, gender, smoking status, and use of oral or inhaled steroids were recorded for a cohort of 199 patients whose spontaneously expectorated sputum samples were submitted to a Cytology Laboratory in Spain between January 2005 and December 2006. Slides were scanned for protozoa under light microscopy and scanned for monocytes/small macrophages highlighted by immunocytochemistry (CD68 monoclonal antibody). One hundred ninety-one patients provided adequate sputum samples, of whom 70 had protozoa in their sputum. There was a strong relationship between the presence of protozoa and monocytes/small macrophages identified under light microscopy (P < 0.001). A binary logistic regression model also indicated a relationship between protozoa and both smoking status and steroid use. The diagnoses in those with protozoa included infection (including tuberculosis), chronic obstructive pulmonary disease (COPD), lung fibrosis, asthma, chronic liver disease, immunosuppression, cancer, pancreatic or renal disease, heart failure, and AIDS. The identified association between protozoa and monocytes/small macrophages in sputum suggests an immune response and warrants further investigation to clarify whether or not these organisms have any pathological significance in this wide range of conditions. Copyright © 2011 Wiley Periodicals, Inc.

Colour of sputum is a marker for bacterial colonisation in chronic obstructive pulmonary disease.

PubMed

Miravitlles, Marc; Marín, Alicia; Monsó, Eduard; Vilà, Sara; de la Roza, Cristian; Hervás, Ramona; Esquinas, Cristina; García, Marian; Millares, Laura; Morera, Josep; Torres, Antoni

2010-05-14

Bacterial colonisation in chronic obstructive pulmonary disease (COPD) contributes to airway inflammation and modulates exacerbations. We assessed risk factors for bacterial colonisation in COPD. Patients with stable COPD consecutively recruited over 1 year gave consent to provide a sputum sample for microbiologic analysis. Bronchial colonisation by potentially pathogenic microorganisms (PPMs) was defined as the isolation of PPMs at concentrations of > or =102 colony-forming units (CFU)/mL on quantitative bacterial culture. Colonised patients were divided into high (>105 CFU/mL) or low (<105 CFU/mL) bacterial load. A total of 119 patients (92.5% men, mean age 68 years, mean forced expiratory volume in one second [FEV1] [% predicted] 46.4%) were evaluated. Bacterial colonisation was demonstrated in 58 (48.7%) patients. Patients with and without bacterial colonisation showed significant differences in smoking history, cough, dyspnoea, COPD exacerbations and hospitalisations in the previous year, and sputum colour. Thirty-six patients (62% of those colonised) had a high bacterial load. More than 80% of the sputum samples with a dark yellow or greenish colour yielded PPMs in culture. In contrast, only 5.9% of white and 44.7% of light yellow sputum samples were positive (P < 0.001). Multivariate analysis showed an increased degree of dyspnoea (odds ratio [OR] = 2.63, 95% confidence interval [CI] 1.53-5.09, P = 0.004) and a darker sputum colour (OR = 4.11, 95% CI 2.30-7.29, P < 0.001) as factors associated with the presence of PPMs in sputum. Almost half of our population of ambulatory moderate to very severe COPD patients were colonised with PPMs. Patients colonised present more severe dyspnoea, and a darker colour of sputum allows identification of individuals more likely to be colonised.

Comparing the Yield of Nasopharyngeal Swabs, Nasal Aspirates, and Induced Sputum for Detection of Bordetella pertussis in Hospitalized Infants.

PubMed

Nunes, Marta C; Soofie, Nasiha; Downs, Sarah; Tebeila, Naume; Mudau, Azwi; de Gouveia, Linda; Madhi, Shabir A

2016-12-01

 Advances in molecular laboratory techniques are changing the landscape of Bordetella pertussis illness diagnosis. Polymerase chain reaction (PCR) assays have greatly improved the sensitivity detection and the turnaround time to diagnosis compared to culture. Moreover, different respiratory specimens, such as flocked nasopharyngeal swabs (NPSs), nasopharyngeal aspirates (NPAs), and induced sputum, have been used for B. pertussis detection, although there is limited head-to-head comparison to evaluating the PCR yield from the 3 sampling methods.  Hospitalized infants <6 months of age who fulfilled a broad syndromic criteria of respiratory illness were tested for B. pertussis infection by PCR on paired NPSs and NPAs; or paired NPSs and induced sputum. An exploratory analysis of B. pertussis culture was performed on induced sputum specimens and in a subset of NPSs.  From November 2014 to May 2015, 484 infants with paired NPSs and NPAs were tested; 15 (3.1%) PCR-confirmed pertussis cases were identified, 13 of which were PCR positive on both samples, while 1 each were positive only on NPS or NPA. From March to October 2015, 320 infants had NPSs and induced sputum collected, and 11 (3.4%) pertussis cases were identified by PCR, including 8 (72.7%) positive on both samples, 1 (9.1%) only positive on NPS, and 2 (18.2%) only positive on induced sputum. The 3 types of specimens had similar negative predictive value >99% and sensitivity >83%. Compared to PCR, culture sensitivity was 60% in induced sputum and 40% in NPSs.  Flocked nasopharyngeal swabs, nasopharyngeal aspirates, and induced sputum performed similarly for the detection of B. pertussis infection in young infants by PCR. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

Comparing the Yield of Nasopharyngeal Swabs, Nasal Aspirates, and Induced Sputum for Detection of Bordetella pertussis in Hospitalized Infants

PubMed Central

Nunes, Marta C.; Soofie, Nasiha; Downs, Sarah; Tebeila, Naume; Mudau, Azwi; de Gouveia, Linda; Madhi, Shabir A.

2016-01-01

Background. Advances in molecular laboratory techniques are changing the landscape of Bordetella pertussis illness diagnosis. Polymerase chain reaction (PCR) assays have greatly improved the sensitivity detection and the turnaround time to diagnosis compared to culture. Moreover, different respiratory specimens, such as flocked nasopharyngeal swabs (NPSs), nasopharyngeal aspirates (NPAs), and induced sputum, have been used for B. pertussis detection, although there is limited head-to-head comparison to evaluating the PCR yield from the 3 sampling methods. Methods. Hospitalized infants <6 months of age who fulfilled a broad syndromic criteria of respiratory illness were tested for B. pertussis infection by PCR on paired NPSs and NPAs; or paired NPSs and induced sputum. An exploratory analysis of B. pertussis culture was performed on induced sputum specimens and in a subset of NPSs. Results. From November 2014 to May 2015, 484 infants with paired NPSs and NPAs were tested; 15 (3.1%) PCR-confirmed pertussis cases were identified, 13 of which were PCR positive on both samples, while 1 each were positive only on NPS or NPA. From March to October 2015, 320 infants had NPSs and induced sputum collected, and 11 (3.4%) pertussis cases were identified by PCR, including 8 (72.7%) positive on both samples, 1 (9.1%) only positive on NPS, and 2 (18.2%) only positive on induced sputum. The 3 types of specimens had similar negative predictive value >99% and sensitivity >83%. Compared to PCR, culture sensitivity was 60% in induced sputum and 40% in NPSs. Conclusions. Flocked nasopharyngeal swabs, nasopharyngeal aspirates, and induced sputum performed similarly for the detection of B. pertussis infection in young infants by PCR. PMID:27838671

Genetically and Phenotypically Distinct Pseudomonas aeruginosa Cystic Fibrosis Isolates Share a Core Proteomic Signature

PubMed Central

Penesyan, Anahit; Kumar, Sheemal S.; Kamath, Karthik; Shathili, Abdulrahman M.; Venkatakrishnan, Vignesh; Krisp, Christoph; Packer, Nicolle H.; Molloy, Mark P.; Paulsen, Ian T.

2015-01-01

The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of “ready-made” nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such extrapolations may be fraught with peril. PMID:26431321

Secretome of transmissible Pseudomonas aeruginosa AES-1R grown in a cystic fibrosis lung-like environment.

PubMed

Scott, Nichollas E; Hare, Nathan J; White, Melanie Y; Manos, Jim; Cordwell, Stuart J

2013-12-06

Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.

Antibiotic Management of Lung Infections in Cystic Fibrosis. II. Nontuberculous Mycobacteria, Anaerobic Bacteria, and Fungi

PubMed Central

Aksamit, Timothy R.; Chotirmall, Sanjay H.; Dasenbrook, Elliott C.; Elborn, J. Stuart; LiPuma, John J.; Ranganathan, Sarath C.; Waters, Valerie J.; Ratjen, Felix A.

2014-01-01

Airway infections are a key component of cystic fibrosis (CF) lung disease. Whereas the approach to common pathogens such as Pseudomonas aeruginosa is guided by a significant body of evidence, other infections often pose a considerable challenge to treating physicians. In Part I of this series on the antibiotic management of difficult lung infections, we discussed bacterial organisms including methicillin-resistant Staphylococcus aureus, gram-negative bacterial infections, and treatment of multiple bacterial pathogens. Here, we summarize the approach to infections with nontuberculous mycobacteria, anaerobic bacteria, and fungi. Nontuberculous mycobacteria can significantly impact the course of lung disease in patients with CF, but differentiation between colonization and infection is difficult clinically as coinfection with other micro-organisms is common. Treatment consists of different classes of antibiotics, varies in intensity, and is best guided by a team of specialized clinicians and microbiologists. The ability of anaerobic bacteria to contribute to CF lung disease is less clear, even though clinical relevance has been reported in individual patients. Anaerobes detected in CF sputum are often resistant to multiple drugs, and treatment has not yet been shown to positively affect patient outcome. Fungi have gained significant interest as potential CF pathogens. Although the role of Candida is largely unclear, there is mounting evidence that Scedosporium species and Aspergillus fumigatus, beyond the classical presentation of allergic bronchopulmonary aspergillosis, can be relevant in patients with CF and treatment should be considered. At present, however there remains limited information on how best to select patients who could benefit from antifungal therapy. PMID:25167882

Antibiotic management of lung infections in cystic fibrosis. II. Nontuberculous mycobacteria, anaerobic bacteria, and fungi.

PubMed

Chmiel, James F; Aksamit, Timothy R; Chotirmall, Sanjay H; Dasenbrook, Elliott C; Elborn, J Stuart; LiPuma, John J; Ranganathan, Sarath C; Waters, Valerie J; Ratjen, Felix A

2014-10-01

Airway infections are a key component of cystic fibrosis (CF) lung disease. Whereas the approach to common pathogens such as Pseudomonas aeruginosa is guided by a significant body of evidence, other infections often pose a considerable challenge to treating physicians. In Part I of this series on the antibiotic management of difficult lung infections, we discussed bacterial organisms including methicillin-resistant Staphylococcus aureus, gram-negative bacterial infections, and treatment of multiple bacterial pathogens. Here, we summarize the approach to infections with nontuberculous mycobacteria, anaerobic bacteria, and fungi. Nontuberculous mycobacteria can significantly impact the course of lung disease in patients with CF, but differentiation between colonization and infection is difficult clinically as coinfection with other micro-organisms is common. Treatment consists of different classes of antibiotics, varies in intensity, and is best guided by a team of specialized clinicians and microbiologists. The ability of anaerobic bacteria to contribute to CF lung disease is less clear, even though clinical relevance has been reported in individual patients. Anaerobes detected in CF sputum are often resistant to multiple drugs, and treatment has not yet been shown to positively affect patient outcome. Fungi have gained significant interest as potential CF pathogens. Although the role of Candida is largely unclear, there is mounting evidence that Scedosporium species and Aspergillus fumigatus, beyond the classical presentation of allergic bronchopulmonary aspergillosis, can be relevant in patients with CF and treatment should be considered. At present, however there remains limited information on how best to select patients who could benefit from antifungal therapy.

Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling.

PubMed

Pérez-Lago, L; Palacios, J J; Herranz, M; Ruiz Serrano, M J; Bouza, E; García-de-Viedma, D

2015-02-01

The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit-variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Educational intervention for collecting sputum for tuberculosis: a quasi-experimental study 1

PubMed Central

Sicsú, Amélia Nunes; Salem, Julia Ignez; Fujimoto, Luciana Botinelly Mendonça; Gonzales, Roxana Isabel Cardozo; Cardoso, Maria do Socorro de Lucena; Palha, Pedro Fredemir

2016-01-01

Abstract Objective: to evaluate the quality of the sputum sample before and after the Nursing guidance to patients. Methods: this is a quasi-experimental research design, single group type, before and after, non-randomized study. The study enrolled patients with suspected pulmonary tuberculosis, respiratory symptomatic patients for over three weeks, aged over 18 years, of both genders and without tuberculosis history in the last two years. The educational intervention consisted of individualized guidance on the collection of sputum sample, which was based on the guidelines of the Ministry of Health of Brazil and on the explanatory folder delivery. Results: in this study participated 138 patients with suspected pulmonary tuberculosis. The results showed significant increase of the samples with purulent particles, volume greater than 5 mL and increased rate of patients diagnosed with tuberculosis, after the educational intervention. Conclusion: it was shown that after the educational intervention, it was observed sputum samples with better quality, with satisfactory aspect and volume for the effectiveness of the bacilloscopic examination. PMID:27276015

Neutrophil extracellular trap formation and extracellular DNA in sputum of stable COPD patients.

PubMed

Pedersen, Frauke; Marwitz, Sebastian; Holz, Olaf; Kirsten, Anne; Bahmer, Thomas; Waschki, Benjamin; Magnussen, Helgo; Rabe, Klaus F; Goldmann, Torsten; Uddin, Mohib; Watz, Henrik

2015-10-01

Chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic airway inflammation. Neutrophil extracellular trap (NET) formation - a meshwork of neutrophil DNA components and neutrophil enzymes are involved in innate immunity and inflammation. Little is known about the presence of these structures in induced sputum from stable COPD patients. Induced sputum samples of 23 COPD patients and 10 healthy controls were collected. Sputum cells were harvested, cultivated and stained for NET components. Extracellular DNA was quantified using a NanoDrop 2000 spectrophotometer. NET formation was markedly upregulated in COPD sputum compared with healthy controls, irrespective of sputum purulence or smoking status. NET formation was associated with significantly higher concentration of extracellular DNA in sputum supernatant (484 ng/μl in COPD versus 268 ng/μl in controls, p = 0.013). Log-transformed extracellular DNA correlated with log-transformed absolute neutrophil numbers in sputum (r = 0.60; p < 0.001) and airway obstruction (r = -0.43; p = 0.013). NET formation associated with higher concentrations of extracellular DNA may be a pathobiological feature of COPD-derived sputum neutrophils. Copyright © 2015 Elsevier Ltd. All rights reserved.

Positive pressure--analysing the effect of the addition of non-invasive ventilation (NIV) to home airway clearance techniques (ACT) in adult cystic fibrosis (CF) patients.

PubMed

Stanford, Gemma; Parrott, Helen; Bilton, Diana; Agent, Penny

2015-05-01

There is no published literature on the frequency of use of non-invasive ventilation (NIV) with airway clearance techniques (ACT) throughout the cystic fibrosis (CF) population; 3.9% (191 people of 5062 registered) of the United Kingdom CF population older than 16 years are reported to use NIV in registry data; however, it is not specified if this is for ACT or respiratory failure. Using NIV with ACT decreases work of breathing and fatigue during in-patient admissions for CF patients. We hypothesised these effects could be replicated at home, potentially reducing hospital admissions. Fourteen adult patients with CF scored ease of clearance and breathlessness with ACT before and after addition of NIV to normal ACT routine using a visual analog scale. Patient views on NIV with ACT were collected via a structured interview. Number of home intravenous (IV) antibiotic courses and days in hospital was collected for one year pre- and post-NIV provision. Patients reported statistically significant improvements in ease of clearance (p = 0.011) and reduced breathlessness during ACT using NIV (p = 0.011). Structured interview results indicated patient reports of sputum clearance improved. In-patient days were lower, while home IV days were higher after NIV was set up, although not statistically significant. This study is limited by small numbers; however, trends towards less hospital admissions and greater patient ease while using NIV with ACT warrant further investigation.

Comparison of Microbiomes from Different Niches of Upper and Lower Airways in Children and Adolescents with Cystic Fibrosis

PubMed Central

Boutin, Sébastien; Graeber, Simon Y.; Weitnauer, Michael; Panitz, Jessica; Stahl, Mirjam; Clausznitzer, Diana; Kaderali, Lars; Einarsson, Gisli; Tunney, Michael M.; Elborn, J. Stuart

2015-01-01

Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both α and β-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible. PMID:25629612

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography–mass spectrometry

PubMed Central

Nicoara, Simona C.; Turner, Nicholas W.; Minnikin, David E.; Lee, Oona Y.-C.; O'Sullivan, Denise M.; McNerney, Ruth; Mutetwa, Reggie; Corbett, Liz E.; Morgan, Geraint H.

2015-01-01

A proof of principle gas chromatography–mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36–68% for PDIMs, allowing some superior detection of the target analytes. PMID:25728371

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kornberg, H.A.; Cline, J.F.

Twenty-four sputum samples were taken from persons working in the vicinity of separation stacks from which radioactive particles are discharged. No evidence of specs'' were sound by radioautography of the sputum plates. Six samples were reset for longer exposure times in an attempt to detect particles of very low activity. Negative results were obtained. 1 tab.

Rapid microbiological screening for tuberculosis in HIV-positive patients on the first day of acute hospital admission by systematic testing of urine samples using Xpert MTB/RIF: a prospective cohort in South Africa.

PubMed

Lawn, Stephen D; Kerkhoff, Andrew D; Burton, Rosie; Schutz, Charlotte; van Wyk, Gavin; Vogt, Monica; Pahlana, Pearl; Nicol, Mark P; Meintjes, Graeme

2015-08-14

Autopsy studies of HIV/AIDS-related hospital deaths in sub-Saharan Africa reveal frequent failure of pre-mortem diagnosis of tuberculosis (TB), which is found in 34-64 % of adult cadavers. We determined the overall prevalence and predictors of TB among consecutive unselected HIV-positive adults requiring acute hospital admission and the comparative diagnostic yield obtained by screening urine and sputum samples obtained on day 1 of admission with Xpert MTB/RIF (Xpert). To determine overall TB prevalence accurately, comprehensive clinical sampling (sputum, urine, blood plus other relevant samples) was done and TB was defined by detection of Mycobacterium tuberculosis in any sample using Xpert and/or mycobacterial liquid culture. To evaluate a rapid screening strategy, we compared the diagnostic yield of Xpert testing sputum samples and urine samples obtained with assistance from a respiratory study nurse in the first 24 h of admission. Unselected HIV-positive acute adult new medical admissions (n = 427) who were not receiving TB treatment were enrolled irrespective of clinical presentation or symptom profile. From 2,391 cultures and Xpert tests done (mean, 5.6 tests/patient) on 1,745 samples (mean, 4.1 samples/patient), TB was diagnosed in 139 patients (median CD4 cell count, 80 cells/μL). TB prevalence was very high (32.6 %; 95 % CI, 28.1-37.2 %; 139/427). However, patient symptoms and risk factors were poorly predictive for TB. Overall, ≥1 non-respiratory sample(s) tested positive in 115/139 (83 %) of all TB cases, including positive blood cultures in 41/139 (29.5 %) of TB cases. In the first 24 h of admission, sputum (spot and/or induced samples) and urine were obtainable from 37.0 % and 99.5 % of patients, respectively (P <0.001). From these, the proportions of total TB cases (n = 139) that were diagnosed by Xpert testing sputum, urine or both sputum and urine combined within the first 24 h were 39/139 (28.1 %), 89/139 (64.0 %) and 108/139 (77.7 %) cases, respectively (P <0.001). The very high prevalence of active TB and its non-specific presentation strongly suggest the need for routine microbiological screening for TB in all HIV-positive medical admissions in high-burden settings. The incremental diagnostic yield from Xpert testing urine was very high and this strategy might be used to rapidly screen new admissions, especially if sputum is difficult to obtain.

Method for efficient storage and transportation of sputum specimens for molecular testing of tuberculosis.

PubMed

Guio, H; Okayama, H; Ashino, Y; Saitoh, H; Xiao, P; Miki, M; Yoshihara, N; Nakanowatari, S; Hattori, T

2006-08-01

The polymerase chain reaction (PCR) is a highly sensitive method for the detection of Mycobacterium tuberculosis and is available in most countries, though to a lesser extent in rural areas. To amplify M. tuberculosis DNA sequences of sputum spotted on FTA cards and compare them with the results of microscopic examination among culture-positive samples. A total of 102 sputum specimens of TB patients in treatment were spotted on FTA cards and stored at room temperature until DNA analysis. We assessed the IS6110 region of M. tuberculosis. The efficacy of the PCR assay for the direct detection of M. tuberculosis was evaluated and compared with the results of cultures (Middlebrook 7H9 broth) and smears of fresh sputum specimens. We were able to detect 10 fg/microl of mycobacterial DNA even after 6 months in storage. The PCR sensitivity and specificity using the FTA card system were 82% and 96%, while microscopic examination showed 41% and 95%, respectively. The FTA card system for the storage of bacterial DNA from sputum samples should be considered for the molecular diagnosis of tuberculosis. Samples can easily be obtained from geographically isolated populations and shipped by mail for accurate molecular diagnosis.

Sputum colour can identify patients with neutrophilic inflammation in asthma

PubMed Central

Gibson, Peter; Lochrin, Alyssa J; Wood, Lisa; Baines, Katherine J; Simpson, Jodie L

2017-01-01

Introduction Sputum colour is associated with neutrophilic inflammation in chronic bronchitis and chronic obstructive pulmonary disease (COPD). Neutrophilia and sputum expectoration is notable in asthma, but whether sputum colour is associated with and predicts the presence of neutrophilic inflammation in asthma is unknown. The objective of the study is to assess the ability of sputum colour in distinguishing asthma inflammatory phenotypes. Methods Induced sputum samples collected from 271 adults with stable asthma were retrospectively assessed. Sputum colour was determined using the BronkoTest sputum colour chart and correlated to differential cell counts and CXCL-8 concentration. Neutrophilic inflammation was defined as an age-corrected sputum neutrophil proportion (≥61.6% for age 20–40 years; ≥63.2% for age 40–60 and ≥67.2% for age >60 years), whereas neutrophilic bronchitis (NB) was defined as high total cell count (≥5.1×106 cells/mL) plus an increased age-corrected neutrophil proportion. The optimal cut-off for sputum colour to predict neutrophilic inflammation and NB was determined using receiver operator characteristic curve analysis. Results A sputum colour score of ≥3 represented and predicted neutrophilic inflammation with modest accuracy (area under the curve (AUC)=0.64; p<0.001, specificity=78.4%, sensitivity=49.2%). Participants with a sputum colour score of ≥3 had significantly (p<0.05) higher CXCL-8, total cells and neutrophil number and proportion. Sputum colour score was also positively correlated with these factors. Sputum colour score ≥3 predicted NB with reasonably good accuracy (AUC=0.79, p<0.001, specificity=79.3%, sensitivity=70.7%). Conclusions Visual gradation of sputum colour in asthma relates to high total cell count and neutrophilic inflammation. Assessment of sputum colour can identify adults with asthma who are likely to have NB without the need for sputum processing and differential cell count, which may facilitate asthma management. PMID:29071085

Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru

PubMed Central

Vargas, Daniel; García, Luis; Gilman, Robert H; Evans, Carlton; Ticona, Eduardo; Ñavincopa, Marcos; Luo, Robert F; Caviedes, Luz; Hong, Clemens; Escombe, Rod; Moore, David A J

2010-01-01

Sputum induction, bronchoalveolar lavage, or gastric aspiration are often needed to produce adequate diagnostic respiratory samples from people with HIV in whom tuberculosis is suspected. Since these procedures are rarely appropriate in less-developed countries, we compared the performances of a simple string test and the gold-standard sputum induction. 160 HIV-positive adults under investigation for tuberculosis, and 52 asymptomatic HIV-positive control patients underwent the string test followed by sputum induction. The string test detected tuberculosis in 14 patients in whom this disease was suspected; sputum induction detected only eight of them (McNemar's test, p=0·03). These preliminary data suggest that the string test is safe and effective for retrieval of useful clinical specimens for diagnosis of pulmonary tuberculosis, and is at least as sensitive as sputum induction. PMID:15639297

Effect of airway clearance techniques on the efficacy of the sputum induction procedure.

PubMed

Elkins, M R; Lane, T; Goldberg, H; Pagliuso, J; Garske, L A; Hector, E; Marchetto, L; Alison, J A; Bye, P T P

2005-11-01

Sputum induction is used in the early identification of tuberculosis (TB) and pneumocystis infections of the lung. Although manual physiotherapy techniques to clear the airways are often incorporated in the sputum induction procedure, their efficacy in this setting is unknown. This randomised, crossover trial enrolled adults referred for sputum induction for suspected TB and pneumocystis infections of the lung. All participants underwent two sputum induction procedures, inhaling 3% saline via ultrasonic nebuliser. During one randomly allocated procedure, airway clearance techniques (chest wall percussion, vibration, huffing) were incorporated. In total, 59 participants completed the trial. The airway clearance techniques had no significant effect on how the test was tolerated, the volume expectorated or the quality of the sample obtained (assessed by the presence of alveolar macrophages). The techniques did not significantly affect how often the test identified a suspected organism, nor the sensitivity or specificity of sputum induction. In conclusion, the study was unable to demonstrate any effect of airway clearance techniques on the sputum induction procedure. The results provide some justification for not including airway clearance techniques as part of the sputum induction procedure.

The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalization in patients with cystic fibrosis.

PubMed

Amin, Reshma; Dupuis, Annie; Aaron, Shawn D; Ratjen, Felix

2010-01-01

The relevance of Aspergillus fumigatus in patients with cystic fibrosis (CF) not affected by allergic bronchopulmonary aspergillosis is unclear. Our aim was to determine the effect of persistent infection with A fumigatus on pulmonary exacerbations and lung function in children with CF. This was a retrospective cohort study of patients with CF followed at The Hospital for Sick Children from 1999 to 2006. Persistent A fumigatus infection was defined as the presence of two or more positive sputum or bronchoalveolar cultures for A fumigatus in a given year. The primary outcome measure was the annual number of hospitalizations for pulmonary exacerbations. Two hundred thirty patients with CF were included in the analysis. The FEV(1) of patients persistently infected with A fumigatus was 3.61% (P< or =.0001) lower during the study period compared with uninfected patients. There was a significant interaction between A fumigatus and Pseudomonas aeruginosa on lung function (P=.0006). Patients not infected with either organism had the highest pulmonary function. Persistent A fumigatus infection (relative risk [RR]=1.94, P=.0002) and CF-related diabetes (RR=1.64, P=.028) were associated with an increased risk of pulmonary exacerbations requiring hospitalization, whereas there was no increased risk of pulmonary exacerbations among patients with allergic bronchopulmonary aspergillosis (RR=1.02, P=.94). When adjusted for baseline pulmonary function, none of these variables were associated with a significantly increased risk of pulmonary exacerbations, with only chronic A fumigatus infection trending toward significance (RR=1.40, P=.065). Persistent A fumigatus infection is an independent risk factor for hospital admissions in patients with CF.

Biomarker Surrogates Do Not Accurately Predict Sputum Eosinophils and Neutrophils in Asthma

PubMed Central

Hastie, Annette T.; Moore, Wendy C.; Li, Huashi; Rector, Brian M.; Ortega, Victor E.; Pascual, Rodolfo M.; Peters, Stephen P.; Meyers, Deborah A.; Bleecker, Eugene R.

2013-01-01

Background Sputum eosinophils (Eos) are a strong predictor of airway inflammation, exacerbations, and aid asthma management, whereas sputum neutrophils (Neu) indicate a different severe asthma phenotype, potentially less responsive to TH2-targeted therapy. Variables such as blood Eos, total IgE, fractional exhaled nitric oxide (FeNO) or FEV1% predicted, may predict airway Eos, while age, FEV1%predicted, or blood Neu may predict sputum Neu. Availability and ease of measurement are useful characteristics, but accuracy in predicting airway Eos and Neu, individually or combined, is not established. Objectives To determine whether blood Eos, FeNO, and IgE accurately predict sputum eosinophils, and age, FEV1% predicted, and blood Neu accurately predict sputum neutrophils (Neu). Methods Subjects in the Wake Forest Severe Asthma Research Program (N=328) were characterized by blood and sputum cells, healthcare utilization, lung function, FeNO, and IgE. Multiple analytical techniques were utilized. Results Despite significant association with sputum Eos, blood Eos, FeNO and total IgE did not accurately predict sputum Eos, and combinations of these variables failed to improve prediction. Age, FEV1%predicted and blood Neu were similarly unsatisfactory for prediction of sputum Neu. Factor analysis and stepwise selection found FeNO, IgE and FEV1% predicted, but not blood Eos, correctly predicted 69% of sputum Eos

Eradication of early P. aeruginosa infection in children <7 years of age with cystic fibrosis: The early study.

PubMed

Ratjen, Felix; Moeller, Alexander; McKinney, Martha L; Asherova, Irina; Alon, Nipa; Maykut, Robert; Angyalosi, Gerhild

2018-04-20

Antibiotic eradication treatment is the standard-of-care for cystic fibrosis (CF) patients with early Pseudomonas aeruginosa (Pa)-infection; however, evidence from placebo-controlled trials is limited. This double-blind, placebo-controlled trial randomised CF patients <7 years (N = 51) with early Pa-infection to tobramycin inhalation solution (TOBI 300 mg) or placebo (twice daily) for 28 days with an optional cross-over on Day 35. Primary endpoint was proportion of patients having throat swabs/sputum free of Pa on Day 29. On Day 29, 84.6% patients in the TOBI versus 24.0% in the placebo group were Pa-free (p < 0.001). At the end of the cross-over period, 76.0% patients receiving TOBI in the initial 28 days were Pa-free compared to 47.8% receiving placebo initially. Adverse events were consistent with the TOBI safety profile with no differences between TOBI and placebo. TOBI was effective in eradicating early Pa-infection with a favourable safety profile in young CF patients. NCT01082367. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

A new method to improve the clinical evaluation of cystic fibrosis patients by mucus viscoelastic properties.

PubMed

Tomaiuolo, Giovanna; Rusciano, Giulia; Caserta, Sergio; Carciati, Antonio; Carnovale, Vincenzo; Abete, Pasquale; Sasso, Antonio; Guido, Stefano

2014-01-01

In cystic fibrosis (CF) patients airways mucus shows an increased viscoelasticity due to the concentration of high molecular weight components. Such mucus thickening eventually leads to bacterial overgrowth and prevents mucus clearance. The altered rheological behavior of mucus results in chronic lung infection and inflammation, which causes most of the cases of morbidity and mortality, although the cystic fibrosis complications affect other organs as well. Here, we present a quantitative study on the correlation between cystic fibrosis mucus viscoelasticity and patients clinical status. In particular, a new diagnostic parameter based on the correlation between CF sputum viscoelastic properties and the severity of the disease, expressed in terms of FEV1 and bacterial colonization, was developed. By using principal component analysis, we show that the types of colonization and FEV1 classes are significantly correlated to the elastic modulus, and that the latter can be used for CF severity classification with a high predictive efficiency (88%). The data presented here show that the elastic modulus of airways mucus, given the high predictive efficiency, could be used as a new clinical parameter in the prognostic evaluation of cystic fibrosis.

Evaluation of Xpert® MTB/RIF Assay in Induced Sputum and Gastric Lavage Samples from Young Children with Suspected Tuberculosis from the MVA85A TB Vaccine Trial.

PubMed

Bunyasi, Erick Wekesa; Tameris, Michele; Geldenhuys, Hennie; Schmidt, Bey-Marrie; Luabeya, Angelique Kany Kany; Mulenga, Humphrey; Scriba, Thomas J; Hanekom, Willem A; Mahomed, Hassan; McShane, Helen; Hatherill, Mark

2015-01-01

Diagnosis of childhood tuberculosis is limited by the paucibacillary respiratory samples obtained from young children with pulmonary disease. We aimed to compare accuracy of the Xpert® MTB/RIF assay, an automated nucleic acid amplification test, between induced sputum and gastric lavage samples from young children in a tuberculosis endemic setting. We analyzed standardized diagnostic data from HIV negative children younger than four years of age who were investigated for tuberculosis disease near Cape Town, South Africa [2009-2012]. Two paired, consecutive induced sputa and early morning gastric lavage samples were obtained from children with suspected tuberculosis. Samples underwent Mycobacterial Growth Indicator Tube [MGIT] culture and Xpert MTB/RIF assay. We compared diagnostic yield across samples using the two-sample test of proportions and McNemar's χ2 test; and Wilson's score method to calculate sensitivity and specificity. 1,020 children were evaluated for tuberculosis during 1,214 admission episodes. Not all children had 4 samples collected. 57 of 4,463[1.3%] and 26 of 4,606[0.6%] samples tested positive for Mycobacterium tuberculosis on MGIT culture and Xpert MTB/RIF assay respectively. 27 of 2,198[1.2%] and 40 of 2,183[1.8%] samples tested positive [on either Xpert MTB/RIF assay or MGIT culture] on induced sputum and gastric lavage samples, respectively. 19/1,028[1.8%] and 33/1,017[3.2%] admission episodes yielded a positive MGIT culture or Xpert MTB/RIF assay from induced sputum and gastric lavage, respectively. Sensitivity of Xpert MTB/RIF assay was 8/30[26.7%; 95% CI: 14.2-44.4] for two induced sputum samples and 7/31[22.6%; 11.4-39.8] [p = 0.711] for two gastric lavage samples. Corresponding specificity was 893/893[100%;99.6-100] and 885/890[99.4%;98.7-99.8] respectively [p = 0.025]. Sensitivity of Xpert MTB/RIF assay was low, compared to MGIT culture, but diagnostic performance of Xpert MTB/RIF did not differ sufficiently between induced sputum and gastric lavage to justify selection of one sampling method over the other, in young children with suspected pulmonary TB. ClinicalTrials.gov NCT00953927.

Reduced Bacterial Colony Count of Anaerobic Bacteria Is Associated with a Worsening in Lung Clearance Index and Inflammation in Cystic Fibrosis

PubMed Central

Bradley, Judy M.; Johnston, Elinor; McGrath, Stephanie; McIlreavey, Leanne; Rowan, Stephen; Reid, Alastair; Bradbury, Ian; Einarsson, Gisli

2015-01-01

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung. PMID:25992575

Reduced bacterial colony count of anaerobic bacteria is associated with a worsening in lung clearance index and inflammation in cystic fibrosis.

PubMed

O'Neill, Katherine; Bradley, Judy M; Johnston, Elinor; McGrath, Stephanie; McIlreavey, Leanne; Rowan, Stephen; Reid, Alastair; Bradbury, Ian; Einarsson, Gisli; Elborn, J Stuart; Tunney, Michael M

2015-01-01

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

The effect of the weather on pulmonary exacerbations and viral infections among adults with cystic fibrosis.

PubMed

Flight, W G; Bright-Thomas, R J; Sarran, C; Mutton, K J; Morris, J; Webb, A K; Jones, A M

2014-11-01

The effect of changes in the weather on the respiratory health of patients with cystic fibrosis (CF) is unclear. We conducted a prospective study to determine the impact of climate and season on the incidence of viral respiratory infections (VRI) and pulmonary exacerbations (PEx) among adults with CF. Between December 2010 and April 2012, 98 adults with CF were followed for 12 months. Polymerase chain reaction assays for nine viruses were performed on sputum, nose and throat swabs every 2 months and additionally at onset of PEx. Hourly temperature and relative humidity measurements were recorded throughout the study. Statistical analysis utilized generalized estimating equation (GEE) models. Pre-specified criteria for VRI and PEx were met at 29% and 37% of visits, respectively. Rhinovirus accounted for 72% of identified viruses. Incidence of rhinovirus peaked in autumn while non-rhinovirus VRI peaked in winter. Rhinovirus was associated with increased mean temperatures (OR 1.07; p = 0.001), while non-rhinovirus VRI was associated with lower mean temperatures (OR 0.87; p < 0.001). PEx occurred frequently throughout the study with no clear seasonal pattern observed. There was no significant association between climate variables and the incidence of either PEx or antibiotic prescription. There is a seasonal pattern to VRI in adults with CF. The incidence of VRI but not PEx is associated with changes in ambient temperature.

[The use of 16S rDNA sequencing in species diversity analysis for sputum of patients with ventilator-associated pneumonia].

PubMed

Yang, Xiaojun; Wang, Xiaohong; Liang, Zhijuan; Zhang, Xiaoya; Wang, Yanbo; Wang, Zhenhai

2014-05-01

To study the species and amount of bacteria in sputum of patients with ventilator-associated pneumonia (VAP) by using 16S rDNA sequencing analysis, and to explore the new method for etiologic diagnosis of VAP. Bronchoalveolar lavage sputum samples were collected from 31 patients with VAP. Bacterial DNA of the samples were extracted and identified by polymerase chain reaction (PCR). At the same time, sputum specimens were processed for routine bacterial culture. The high flux sequencing experiment was conducted on PCR positive samples with 16S rDNA macro genome sequencing technology, and sequencing results were analyzed using bioinformatics, then the results between the sequencing and bacteria culture were compared. (1) 550 bp of specific DNA sequences were amplified in sputum specimens from 27 cases of the 31 patients with VAP, and they were used for sequencing analysis. 103 856 sequences were obtained from those sputum specimens using 16S rDNA sequencing, yielding approximately 39 Mb of raw data. Tag sequencing was able to inform genus level in all 27 samples. (2) Alpha-diversity analysis showed that sputum samples of patients with VAP had significantly higher variability and richness in bacterial species (Shannon index values 1.20, Simpson index values 0.48). Rarefaction curve analysis showed that there were more species that were not detected by sequencing from some VAP sputum samples. (3) Analysis of 27 sputum samples with VAP by using 16S rDNA sequences yielded four phyla: namely Acitinobacteria, Bacteroidetes, Firmicutes, Proteobacteria. With genus as a classification, it was found that the dominant species included Streptococcus 88.9% (24/27), Limnohabitans 77.8% (21/27), Acinetobacter 70.4% (19/27), Sphingomonas 63.0% (17/27), Prevotella 63.0% (17/27), Klebsiella 55.6% (15/27), Pseudomonas 55.6% (15/27), Aquabacterium 55.6% (15/27), and Corynebacterium 55.6% (15/27). (4) Pyrophosphate sequencing discovered that Prevotella, Limnohabitans, Aquabacterium, Sphingomonas might not be detected by routine bacteria culture. Among seven species which were identified by both methods, pyrophosphate sequencing yielded higher positive rate than that of ordinary bacteria culture [Streptococcus: 88.9% (24/27) vs. 18.5% (5/27), Klebsiella: 55.6% (15/27) vs. 18.5% (5/27), Acinetobacter: 70.4% (19/27) vs. 37.0% (10/27), Corynebacterium: 55.6% (15/27) vs. 7.4% (2/27), P<0.05 or P<0.01]. Sequencing positive rate was found to increase positive rate for culture of Pseudomonas [55.6% (15/27) vs. 25.9% (7/27), P=0.050]. No significant differences were observed between sequencing and ordinary bacteria culture for detection Staphylococcus [7.4% (2/27) vs. 11.1% (3/27)] and Neisseria bacteria genera [18.5% (5/27) vs. 3.7% (1/27), both P>0.05]. 16S rDNA sequencing analysis confirmed that pathogenic bacteria in sputum of VAP were complicated with multiple drug resistant strains. Compared with routine bacterial culture, pyrophosphate sequencing had higher positive rate in detecting pathogens. 16S rDNA gene sequencing technology may become a new method for etiological diagnosis of VAP.

Performance of OMNIgene•SPUTUM (DNA Genotek) and cetylpyridinium chloride for sputum storage prior to mycobacterial culture.

PubMed

Affolabi, Dissou; Sanoussi, N'Dira; Sossou, Adelaide; Nys, Tom; Bodi, Ousman; Esse, Marius; Houeto, Sabine; Massou, Faridath; de Jong, Bouke C; Rigouts, Leen

2018-05-02

The aim was to assess the performance of both cetylpyridinium chloride (CPC) and OMNIgene•SPUTUM (OMNI) reagents for the maintenance of Mycobacterium tuberculosis viability in sputum prior to recovery by culture. Using 312 sputa, we evaluated the performance of the two reagents using culture on Löwenstein-Jensen medium after sputum storage in CPC or OMNI for up to 28 days. In addition, the viability of M. tuberculosis isolates stored in both reagents was assessed. The contamination rates for freshly processed samples and those stored in CPC were not statistically different, while the contamination rate for OMNI was significantly lower than that for fresh sputa (P=0.026 for 8 days and P=0.002 for 28 days of storage). The culture positivity for fresh sputa (81.7 %) was similar to that for samples stored in CPC, regardless of the storage time (89.8 % for CPC-8 and 73.0 % for CPC-28). For OMNI-preserved samples, the culture positivity was similar after 8 days of storage (84.2 %), but decreased significantly after 28 days (42.7 %; P<0.0001) compared to fresh sputa, CPC-8, CPC-28 and OMNI-8. There was a significant loss of viability for the H37Rv strain when it was stored in OMNI at room temperature beyond 8 days compared to CPC, but storage at 37 °C decreased recovery from both CPC- and OMNI-stored suspensions. Culture from sputum stored for 8 days at room temperature in OMNI or CPC gave comparable culture positivity rates to culture from fresh sputum, but after 28 days of storage the performance of OMNI decreased significantly compared to CPC.

Investigation of OMNIgene·SPUTUM performance in delayed tuberculosis testing by smear, culture, and Xpert MTB/RIF assays in Uganda.

PubMed

Kelly-Cirino, C D; Musisi, E; Byanyima, P; Kaswabuli, S; Andama, A; Sessolo, A; Sanyu, I; Zawedde, J; Curry, P S; Huang, L

2017-06-01

OMNIgene·SPUTUM (OM-S) is a sample transport reagent designed to work with all tuberculosis diagnostics while eliminating the need for cold chain. OM-S-treated sputum samples were assayed in several tests after multiday holds. Raw sputa from 100 patients underwent direct smear microscopy, were manually split and assigned to the OM-S group [OM-S added at collection (no other processing required) and tested after 0- to 5-day holds at room temperature] or standard-of-care (SOC) group (NaOH/N-acetyl l-cysteine decontamination, all tested on day of collection). Concentrated smear microscopy, Lowenstein Jensen (LJ) culture, and mycobacteria growth indicator tube (MGIT) culture were performed. For patients with negative direct smear, a second sample was split, with SOC (raw sputum) and OM-S portions (sediment) tested in the Xpert MTB/RIF (Xpert) assay. OM-S group and SOC group results were strongly concordant on all four tests [range, 89% (MGIT)-97% (Xpert)]. OM-S MGIT, LJ, and Xpert tests were in statistical agreement with SOC MGIT as reference. OM-S specimens had lower culture contamination rates (3% vs. 10% LJ; 2% vs. 5% MGIT) but required, on average, 5.6 additional days to become MGIT-positive. The findings suggest that samples held/transported in OM-S are compatible with smear microscopy, LJ or MGIT culture, and Xpert, and perform comparably to fresh sputum samples. Larger feasibility studies are warranted. Copyright © 2017. Published by Elsevier Ltd.

φ(2)GFP10, a high-intensity fluorophage, enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples.

PubMed

Jain, Paras; Hartman, Travis E; Eisenberg, Nell; O'Donnell, Max R; Kriakov, Jordan; Govender, Karnishree; Makume, Mantha; Thaler, David S; Hatfull, Graham F; Sturm, A Willem; Larsen, Michelle H; Moodley, Preshnie; Jacobs, William R

2012-04-01

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (φ(2)GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing φ(2)GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ(2)GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of φ(2)GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with φ(2)GFP10. Fluorophage φ(2)GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.

Comparison of loop-mediated isothermal amplification assay and smear microscopy with culture for the diagnostic accuracy of tuberculosis.

PubMed

Gelaw, Baye; Shiferaw, Yitayal; Alemayehu, Marta; Bashaw, Abate Assefa

2017-01-17

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading causes of death from infectious diseases worldwide. Sputum smear microscopy remains the most widely available pulmonary TB diagnostic tool particularly in resource limited settings. A highly sensitive diagnostic with minimal infrastructure, cost and training is required. Hence, we assessed the diagnostic performance of Loop-mediated isothermal amplification (LAMP) assay in detecting M.tuberculosis infection in sputum sample compared to LED fluorescent smear microscopy and culture. A cross-sectional study was conducted at the University of Gondar Hospital from June 01, 2015 to August 30, 2015. Pulmonary TB diagnosis using sputum LED fluorescence smear microscopy, TB-LAMP assay and culture were done. A descriptive analysis was used to determine demographic characteristics of the study participants. Analysis of sensitivity and specificity for smear microscopy and TB-LAMP compared with culture as a reference test was performed. Cohen's kappa was calculated as a measure of agreement between the tests. A total of 78 pulmonary presumptive TB patients sputum sample were analyzed. The overall sensitivity and specificity of LAMP were 75 and 98%, respectively. Among smear negative sputum samples, 33.3% sensitivity and 100% specificity of LAMP were observed. Smear microscopy showed 78.6% sensitivity and 98% specificity. LAMP and smear in series had sensitivity of 67.8% and specificity of 100%. LAMP and smear in parallel had sensitivity of 85.7% and specificity of 96%. The agreement between LAMP and fluorescent smear microscopy tests was very good (κ = 0.83, P-value ≤0.0001). TB-LAMP showed similar specificity but a slightly lower sensitivity with LED fluorescence microscopy. The specificity of LAMP and smear microscopy in series was high. The sensitivity of LAMP was insufficient for smear negative sputum samples.

Increased detection of smear-negative pulmonary tuberculosis by GeneXpert MTB/RIF® assay after bleach concentration.

PubMed

Tadesse, Mulualem; Aragaw, Dossegnaw; Rigouts, Leen; Abebe, Gemeda

2016-06-01

The GeneXpert MTB/RIF assay (Xpert) was endorsed as the initial diagnostic tool in people suspected of human immunodeficiency virus-associated or drug-resistant tuberculosis (TB). However, information regarding the performance of Xpert for diagnosing smear-negative TB in high burden settings remains limited. We evaluated the diagnostic accuracy of Xpert and the impact of bleach concentration on the performance of Xpert using smear-negative sputum samples from human immunodeficiency virus-negative patients. One spot and one morning smear-negative sputum samples per patient were examined using Xpert and culture at the Mycobacteriology Research Center of Jimma University, Ethiopia. The sputum culture on both Löwenstein-Jensen and/or Mycobacteria Growth Indicator Tube was the gold-standard. Of 185 smear-negative presumptive pulmonary TB cases, 19 (10.3%) had culture-proven TB. The sensitivity of Xpert on spot and morning sputum was similar (63.2%). Testing two specimens per patient insignificantly increased the sensitivity of Xpert. Bleach concentration and pelleting improved the sensitivity of Xpert over unprocessed sputum in paired samples (73.8% vs. 63.2%) without affecting the specificity (95%). Bleach concentration and pelleting allowed an additional seven cases of TB (missed on the first and second direct Xperts) to be detected, five of which were from culture-negative cases. Testing of a single sputum sample by Xpert can reach reasonable sensitivity and results would be available on the same day, avoiding loss of patients and treatment delay. The sensitivity of Xpert was improved after bleach concentration and pelleting, although its added value needs further study on a larger scale. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells

PubMed Central

Moreau-Marquis, Sophie; Coutermarsh, Bonita; Stanton, Bruce A.

2015-01-01

Objectives Chelating iron may be a promising new therapy to eliminate Pseudomonas aeruginosa biofilms in the lungs of cystic fibrosis (CF) patients. Here, we investigate whether ALX-109 [a defined combination of an investigational drug containing lactoferrin (an iron-binding glycoprotein) and hypothiocyanite (a bactericidal agent)], alone and in combination with tobramycin or aztreonam, reduces P. aeruginosa biofilms grown on human CF airway epithelial cells. Methods P. aeruginosa (PAO1 and six clinical isolates of Pseudomonas) biofilms grown at the apical surface of confluent monolayers of CF airway epithelial cells were treated with ALX-109, either alone or in combination with tobramycin or aztreonam. Bacterial cfu remaining after treatment were determined by plate counting. Results ALX-109 alone reduced PAO1 biofilm formation, but had no effect on established biofilms. ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit PAO1 biofilm formation and to reduce established PAO1 biofilms. ALX-109 and tobramycin were additive in disrupting established biofilms formed by six clinical isolates of P. aeruginosa obtained from the sputum of CF patients. Mucoid P. aeruginosa isolates were most susceptible to the combination of ALX-109 and tobramycin. In addition, ALX-109 also enhanced the ability of aztreonam to reduce established PAO1 biofilms. Conclusions Inhalation therapy combining hypothiocyanite and lactoferrin with TOBI® (tobramycin) or Cayston® (aztreonam) may be beneficial to CF patients by decreasing the airway bacterial burden of P. aeruginosa. PMID:25213272

Global impact of bronchiectasis and cystic fibrosis

PubMed Central

Redondo, Margarida; Keyt, Holly; Dhar, Raja

2016-01-01

Educational aims To recognise the clinical and radiological presentation of the spectrum of diseases associated with bronchiectasis. To understand variation in the aetiology, microbiology and burden of bronchiectasis and cystic fibrosis across different global healthcare systems. Bronchiectasis is the term used to refer to dilatation of the bronchi that is usually permanent and is associated with a clinical syndrome of cough, sputum production and recurrent respiratory infections. It can be caused by a range of inherited and acquired disorders, or may be idiopathic in nature. The most well recognised inherited disorder in Western countries is cystic fibrosis (CF), an autosomal recessive condition that leads to progressive bronchiectasis, bacterial infection and premature mortality. Both bronchiectasis due to CF and bronchiectasis due to other conditions are placing an increasing burden on healthcare systems internationally. Treatments for CF are becoming more effective leading to more adult patients with complex healthcare needs. Bronchiectasis not due to CF is becoming increasingly recognised, particularly in the elderly population. Recognition is important and can lead to identification of the underlying cause, appropriate treatment and improved quality of life. The disease is highly diverse in its presentation, requiring all respiratory physicians to have knowledge of the different “bronchiectasis syndromes”. The most common aetiologies and presenting syndromes vary depending on geography, with nontuberculous mycobacterial disease predominating in some parts of North America, post-infectious and idiopathic disease predominating in Western Europe, and post-tuberculosis bronchiectasis dominating in South Asia and Eastern Europe. Ongoing global collaborative studies will greatly advance our understanding of the international impact of bronchiectasis and CF. PMID:28210295

Predictive Detection of Tuberculosis using Electronic Nose Technology

NASA Astrophysics Data System (ADS)

Gibson, Tim; Kolk, Arend; Reither, Klaus; Kuipers, Sjoukje; Hallam, Viv; Chandler, Rob; Dutta, Ritaban; Maboko, Leonard; Jung, Jutta; Klatser, Paul

2009-05-01

The adaptation and use of a Bloodhound® ST214 electronic nose to rapidly detect TB in sputum samples has been discussed in the past, with some promising results being obtained in 2007. Some of the specific VOC's associated with Mycobacteria tuberculosis organisms are now being discovered and a paper was published in 2008, but the method of predicting the presence of TB in sputum samples using the VOC biomarkers has yet to be fully optimised. Nevertheless, with emphasis on the sampling techniques and with new data processing techniques to obtain consistent results progress is being made Sensitivity and specificity levels for field detection of TB have been set by WHO at a minimum level of 85% and 95% respectively, and the e-nose technique is working towards these figures. In a series of experiments carried out in Mbeya, Tanzania, Africa, data from a full 5 days of sampling was combined giving a total of 248 sputum samples analysed. From the data obtained we can report results that show specificities and sensitivities in the 70-80% region when actually predicting the presence of TB in unknown sputum samples. The results are a further step forward in the rapid detection of TB in the clinics in developing countries and show continued promise for future development of an optimised instrument for TB prediction.

Study of Colour Model for Segmenting Mycobacterium Tuberculosis in Sputum Images

NASA Astrophysics Data System (ADS)

Kurniawardhani, A.; Kurniawan, R.; Muhimmah, I.; Kusumadewi, S.

2018-03-01

One of method to diagnose Tuberculosis (TB) disease is sputum test. The presence and number of Mycobacterium tuberculosis (MTB) in sputum are identified. The presence of MTB can be seen under light microscope. Before investigating through stained light microscope, the sputum samples are stained using Ziehl-Neelsen (ZN) stain technique. Because there is no standard procedure in staining, the appearance of sputum samples may vary either in background colour or contrast level. It increases the difficulty in segmentation stage of automatic MTB identification. Thus, this study investigated the colour models to look for colour channels of colour model that can segment MTB well in different stained conditions. The colour models will be investigated are each channel in RGB, HSV, CIELAB, YCbCr, and C-Y colour model and the clustering algorithm used is k-Means. The sputum image dataset used in this study is obtained from community health clinic in a district in Indonesia. The size of each image was set to 1600x1200 pixels which is having variation in number of MTB, background colour, and contrast level. The experiment result indicates that in all image conditions, blue, hue, Cr, and Ry colour channel can be used to segment MTB in one cluster well.

Reduced detection by Ziehl-Neelsen method of acid-fast bacilli in sputum samples preserved in cetylpyridinium chloride solution.

PubMed

Selvakumar, N; Sudhamathi, S; Duraipandian, M; Frieden, T R; Narayanan, P R

2004-02-01

Twelve health facilities implementing the DOTS strategy, and the Tuberculosis Research Centre (TRC), Chennai, India. To determine the detection rates using Ziehl-Neelsen (ZN) and auramine-phenol to stain acid-fast bacilli (AFB) in sputum samples stored in cetylpyridinium chloride (CPC) solution. Two smears were prepared from each of 988 sputum samples collected in CPC and randomly allocated, one to ZN and the other to auramine-phenol staining. All samples were processed for culture of Mycobacterium tuberculosis. A significantly higher proportion of samples were negative using the ZN method compared to the auramine-phenol method (74.5% vs. 61.8%, McNamara's paired chi2 test; P < 0.001). Among 377 samples that were positive using auramine-phenol, 44% were negative using ZN. There were more culture-positive, smear-negative samples in ZN (52.7%) than in auramine-phenol (30%); the difference attained statistical significance (McNemar's paired chi2 test; P < 0.00004). Using ZN, of the 104 smears made immediately after collection, 52 were positive for AFB, of which only 35 (67.3%) were positive after storage in CPC; the reduction in the number of positive smears attained statistical significance (McNemar's paired chi2 test; P = 0.004). Detection of AFB in sputum samples preserved in CPC is significantly reduced using ZN staining.

Proteomic analysis of serum and sputum analytes distinguishes controlled and poorly controlled asthmatics.

PubMed

Kasaian, M T; Lee, J; Brennan, A; Danto, S I; Black, K E; Fitz, L; Dixon, A E

2018-04-17

A major goal of asthma therapy is to achieve disease control, with maintenance of lung function, reduced need for rescue medication, and prevention of exacerbation. Despite current standard of care, up to 70% of patients with asthma remain poorly controlled. Analysis of serum and sputum biomarkers could offer insights into parameters associated with poor asthma control. To identify signatures as determinants of asthma disease control, we performed proteomics using Olink proximity extension analysis. Up to 3 longitudinal serum samples were collected from 23 controlled and 25 poorly controlled asthmatics. Nine of the controlled and 8 of the poorly controlled subjects also provided 2 longitudinal sputum samples. The study included an additional cohort of 9 subjects whose serum was collected within 48 hours of asthma exacerbation. Two separate pre-defined Proseek Multiplex panels (INF and CVDIII) were run to quantify 181 separate protein analytes in serum and sputum. Panels consisting of 9 markers in serum (CCL19, CCL25, CDCP1, CCL11, FGF21, FGF23, Flt3L, IL-10Rβ, IL-6) and 16 markers in sputum (tPA, KLK6, RETN, ADA, MMP9, Chit1, GRN, PGLYRP1, MPO, HGF, PRTN3, DNER, PI3, Chi3L1, AZU1, and OPG) distinguished controlled and poorly controlled asthmatics. The sputum analytes were consistent with a pattern of neutrophil activation associated with poor asthma control. The serum analyte profile of the exacerbation cohort resembled that of the controlled group rather than that of the poorly controlled asthmatics, possibly reflecting a therapeutic response to systemic corticosteroids. Proteomic profiles in serum and sputum distinguished controlled and poorly controlled asthmatics, and were maintained over time. Findings support a link between sputum neutrophil markers and loss of asthma control. © 2018 John Wiley & Sons Ltd.

The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.

PubMed

Chakravorty, Soumitesh; Simmons, Ann Marie; Rowneki, Mazhgan; Parmar, Heta; Cao, Yuan; Ryan, Jamie; Banada, Padmapriya P; Deshpande, Srinidhi; Shenai, Shubhada; Gall, Alexander; Glass, Jennifer; Krieswirth, Barry; Schumacher, Samuel G; Nabeta, Pamela; Tukvadze, Nestani; Rodrigues, Camilla; Skrahina, Alena; Tagliani, Elisa; Cirillo, Daniela M; Davidow, Amy; Denkinger, Claudia M; Persing, David; Kwiatkowski, Robert; Jones, Martin; Alland, David

2017-08-29

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R. Copyright © 2017 Chakravorty et al.

Assessment of the sensitivity and specificity of Xpert MTB/RIF assay as an early sputum biomarker of response to tuberculosis treatment.

PubMed

Friedrich, Sven O; Rachow, Andrea; Saathoff, Elmar; Singh, Kasha; Mangu, Chacha D; Dawson, Rodney; Phillips, Patrick Pj; Venter, Amour; Bateson, Anna; Boehme, Catharina C; Heinrich, Norbert; Hunt, Robert D; Boeree, Martin J; Zumla, Alimuddin; McHugh, Timothy D; Gillespie, Stephen H; Diacon, Andreas H; Hoelscher, Michael

2013-08-01

An accurate biomarker is urgently needed to monitor the response to treatment in patients with pulmonary tuberculosis. The Xpert MTB/RIF assay is a commercially available real-time PCR that can be used to detect Mycobacterium-tuberculosis-specific DNA sequences in sputum samples. We therefore evaluated this assay with serial sputum samples obtained over 26 weeks from patients undergoing treatment for tuberculosis. We analysed sputum samples from 221 patients with smear-positive tuberculosis enrolled at two sites (Cape Town, South Africa, and Mbeya, Tanzania) of a multicentre randomised clinical trial REMoxTB of antituberculosis treatment on a weekly basis (weeks 0 to 8), then at weeks 12, 17, 22, and 26 after treatment initiation. The Xpert MTB/RIF results over time were compared with the results of standard smear microscopy and culture methods. We obtained and analysed 2741 sputum samples from 221 patients. The reduction in positivity rates with Xpert MTB/RIF were slower than those with the standard methods. At week 8, positive results were obtained for 62 (29%) of 212 sputum samples with smear microscopy, 46 (26%) of 175 with solid culture (Löwenstein-Jensen medium), 77 (42%) of 183 with liquid culture (Bactec MGIT960 system), and 174 (84%) of 207 with Xpert MTB/RIF; at 26 weeks, positive results were obtained for ten (5%) of 199, four (3%) of 157, seven (4%) of 169, and 22 (27%) of 83 sputum samples, respectively. The reduction in detection of quantitative M tuberculosis DNA with Xpert MTB/RIF correlated with smear grades (ρ=-0·74; p<0·0001), solid culture grades (ρ=-0·73; p<0·0001), and time to liquid culture positivity (ρ=0·73; p<0·0001). Compared with the combined binary smear and culture results as a reference standard, the Xpert MTB/RIF assay had high sensitivity (97·0%, 95% CI 95·8-97·9), but poor specificity (48·6%, 45·0-52·2). The poor specificity precludes the use of the Xpert MTB/RIF assay as a biomarker for monitoring tuberculosis treatment, and should not replace standard smear microscopy and culture. Global Alliance for TB Drug Development, Bill & Melinda Gates Foundation, UK Medical Research Council, German Ministry of Science and Technology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reliability of a rapid hematology stain for sputum cytology*

PubMed Central

Gonçalves, Jéssica; Pizzichini, Emilio; Pizzichini, Marcia Margaret Menezes; Steidle, Leila John Marques; Rocha, Cristiane Cinara; Ferreira, Samira Cardoso; Zimmermann, Célia Tânia

2014-01-01

Objective: To determine the reliability of a rapid hematology stain for the cytological analysis of induced sputum samples. Methods: This was a cross-sectional study comparing the standard technique (May-Grünwald-Giemsa stain) with a rapid hematology stain (Diff-Quik). Of the 50 subjects included in the study, 21 had asthma, 19 had COPD, and 10 were healthy (controls). From the induced sputum samples collected, we prepared four slides: two were stained with May-Grünwald-Giemsa, and two were stained with Diff-Quik. The slides were read independently by two trained researchers blinded to the identification of the slides. The reliability for cell counting using the two techniques was evaluated by determining the intraclass correlation coefficients (ICCs) for intraobserver and interobserver agreement. Agreement in the identification of neutrophilic and eosinophilic sputum between the observers and between the stains was evaluated with kappa statistics. Results: In our comparison of the two staining techniques, the ICCs indicated almost perfect interobserver agreement for neutrophil, eosinophil, and macrophage counts (ICC: 0.98-1.00), as well as substantial agreement for lymphocyte counts (ICC: 0.76-0.83). Intraobserver agreement was almost perfect for neutrophil, eosinophil, and macrophage counts (ICC: 0.96-0.99), whereas it was moderate to substantial for lymphocyte counts (ICC = 0.65 and 0.75 for the two observers, respectively). Interobserver agreement for the identification of eosinophilic and neutrophilic sputum using the two techniques ranged from substantial to almost perfect (kappa range: 0.91-1.00). Conclusions: The use of Diff-Quik can be considered a reliable alternative for the processing of sputum samples. PMID:25029648

Value of washed sputum gram stain smear and culture for management of lower respiratory tract infections in children.

PubMed

Cao, Luong Dong; Ishiwada, Naruhiko; Takeda, Nobue; Nigo, Yukiko; Aizawa, Jirou; Kuroki, Haruo; Kohno, Yoichi

2004-02-01

To date, the technique of washed sputum examinations has not been widely used in the clinical management of lower respiratory tract infections in children. A total of 224 sputum samples from 125 pediatric patients with lower respiratory tract infections were collected for washed sputum Gram stain smears and cultures. The results with these methods were compared to find correlation rates. The value of washed sputum cultures was assessed by examining the clinical responses of the patients who received antibiotic therapies instituted on the basis of the sputum culture results. Isolation rates of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus were 22.4%, 9.4%, 4.9%, and 0.4%, respectively. For the prediction of H. influenzae, S. pneumoniae, and M. catarrhalis, the sensitivities of the washed sputum Gram stain smears compared with the culture method were 86.0%, 81.0%, and 90.9%, respectively. The specificities of the washed sputum Gram stain smear technique were 94.8%, 97.5%, and 98.1%, respectively. Overall, the sensitivity and specificity of the washed sputum Gram stain smear method were 85.5% and 87.2%, respectively. S. aureus was isolated from only one specimen; and washed sputum Gram stain smear estimation was correlated with the culture result. On the basis of the washed sputum culture results, appropriate antibiotic therapies were instituted for 93.3% of the patients with acute lower respiratory tract infections. This study suggests that the techniques of washed sputum Gram stain smear and culture are valuable and should be encouraged in clinical practice for the management of lower respiratory tract infections in children.

Reduction in Nontuberculous Mycobacteria at a Tuberculosis Hospital Following a Quality Assurance Intervention

PubMed Central

Kizilbash, Quratulain; Jost, Kenneth; Armitige, Lisa; Griffith, David E; Dunbar, Denise; Seaworth, Barbara

2017-01-01

Abstract Background Non tuberculous mycobacteria (NTM) are widely distributed in soil and water. NTM/Mycobacterium tuberculosis complex (MTBC) mixes may yield positive AFB smears falsely attributed to tuberculosis (TB) and false-resistance profiles for TB due to contaminated diagnostic samples. This as well as isolation of NTM may pose diagnostic and management problems. Texas Center for Infectious Disease (TCID) is a hospital for patients with confirmed TB. After a cluster of isolates of Mycobacterium gordonae was identified, a quality assurance review found inadequate protocols which included eating and drinking prior to collection. Changes made to the sputum collection protocol included reeducation of respiratory therapists and a sterile saline rinse intervention prior to sputum collection. Methods All sputa collected for AFB culture from diagnosed TB patients at TCID from January 1st, 2014 to December 31st, 2014 prior to the intervention and from August 1st, 2016 to January 31st, 2017, the 6 months following the quality assurance intervention were included. Sputum samples were processed at the Texas Department of State and Health Services (DSHS) Laboratory. Results A total of 1,853 sputum samples were processed; 1,288 from 2014 and 565 following the intervention. NTM decreased from 56 (4.3%) to 7 (1.2%) after the quality assurance intervention was instituted for a NTM decrease of 75.0%. M. gordonae decreased by 78.6%. No patients had evidence of NTM disease. Conclusion A breach in sputum collection protocols at TCID accounted for the increase in NTM isolation in 2014, half of which were M. gordonae. The reeducation of respiratory therapy staff and initiation of sterile saline rinse prior to sputum collection resulted in a significant reduction in the overall NTM rate. M. gordonae was isolated only three times following the intervention. At TCID, a location where tap water and bottled water contains NTM, drinking these prior to sputum collection possibly contributed to the cluster for NTM, especially M. gordonae. We recommend rinsing the mouth with sterile saline or water prior to sputum collection to decrease isolation of rarely pathogenic NTM. Disclosures All authors: No reported disclosures.

Nasal lavage, blood or sputum: Which is best for phenotyping asthma?

PubMed

de Farias, Camyla F; Amorim, Maria M F; Dracoulakis, Michel; Caetano, Lilian B; Santoro, Ilka L; Fernandes, Ana L G

2017-05-01

Determination of asthma phenotypes, particularly inflammatory phenotypes, helps guide treatment and management of this heterogeneous disease. Induced sputum cytology has been the gold standard for determination of inflammatory phenotypes, but sputum induction is fairly invasive and technically challenging. Blood and nasal lavage cytology have been suggested as substitutes, but have not been fully verified. The aim of this study is to determine the accuracy of blood and nasal lavage cytometry as indicators of inflammatory phenotypes in asthma. Clinical evaluation, Asthma Control Questionnaire (ACQ) and spirometry were performed for 121 adult asthma patients, and blood, nasal lavage and induced sputum samples were taken. Eosinophils and neutrophils were counted in three samples from each subject. Inflammatory phenotypes (eosinophilic, neutrophilic, mixed and paucicellular) and cells counts were analysed using Venn diagram and receiver operating characteristic (ROC) curve, respectively. ACQ score, spirometry and bronchodilator response did not differ among subjects with different inflammatory phenotypes. Inflammatory phenotypes defined by nasal lavage cytometry were in better concordance than those defined by blood cell counts with phenotypes determined by sputum cytology, and were significantly correlated with sputum phenotypes. For eosinophilia, nasal lavage cytology showed better accuracy than blood cytology (area under the curve (AUC): 0.89 vs 0.65). For all phenotypes, sensitivity and positive and negative predictive power were higher for nasal lavage cytometry than for blood. Blood cell counts gave a high level of false positives for all inflammatory phenotypes. We recommend nasal lavage cytology over blood cell count as a substitute for sputum cytology to identify inflammatory phenotypes in asthma. © 2016 Asian Pacific Society of Respirology.

Guaifenesin has no effect on sputum volume or sputum properties in adolescents and adults with acute respiratory tract infections.

PubMed

Hoffer-Schaefer, Agathe; Rozycki, Henry J; Yopp, Melissa A; Rubin, Bruce K

2014-05-01

Guaifenesin (glyceryl guaiacolate ether [GGE]) has been studied as a cough suppressant and as an expectorant; however, published studies to date have failed to find a consistent benefit. An 8-day multi-center clinical trial was conducted to study the effect of two 600-mg extended-release GGE tablets twice daily for 1 week on cold symptoms, sputum volume, and properties in adolescents and adults with productive cough from an acute respiratory tract infection (RTI). The study enrolled 378 subjects (GGE, n = 188; and placebo, n = 190) who were otherwise healthy and had an RTI for up to 5 days before enrollment. Subjects suffered from at least 2 of 3 symptoms of cough, thickened mucus, and chest congestion. A total of 151 GGE and 144 control subjects completed the full protocol. Single-sputum samples were collected from each subject on days 1, 3, 4, and 8 of the study. The rheology and interfacial tension of sputum were measured, and 24-h collected samples from days 1 and 4 were analyzed for total volume and hydration. Symptoms in both the GGE and placebo groups improved to a similar degree over time. There were no significant differences between the GGE and placebo groups for sputum volume (P = .41), percent solids (P = .69), interfacial tension (P = .88), elasticity (P = .71), viscosity (P = .45), or mechanical impedance (P = .75). The recommended dose of GGE had no measurable effect on sputum volume or properties and is unlikely to be an expectorant or mucolytic when used to treat acute RTI. (ClinicalTrials.gov registration NCT01046136.).

NHRC (Naval Health Research Center) Report for 1979

DTIC Science & Technology

1979-01-01

of cross- validation analyses on an independent sample (n = 336) were also reported and were generally consistent with the foregoing analyses. 79-3...antigens in sputum. Initial sputum samples from patients with pneumococcal p,; Lmonia (᝼ hr antibiotic therapy) were positive for antigen in 37 of 44...the decline was gree’ar for CIE (only 29% of samples positive at three days of therapy) than with coagglutination (61% positive at three days) (pɘ.05

Elastolytic activity of sputum and its relation to purulence and to lung function in patients with bronchiectasis.

PubMed Central

Stockley, R A; Hill, S L; Morrison, H M; Starkie, C M

1984-01-01

Sputum samples from 34 patients with bronchiectasis were assessed subjectively and the results related to objective measurements of elastase activity and albumin content. The results suggest that the macroscopic appearance of the sample is related to the elastase content. 88.7% of the purulent samples but none of the mucoid samples showing elastase activity. The macroscopic appearance was also associated with changes in protein transudation into the secretions. The median sputum: serum albumin concentration ratio was 0.71 X 10(-2) (range 0.22-4.7) in the mucoid samples but was greater in purulent samples (p less than 0.005), with a median value of 1.52 X 10(-2) (range 0.55-12.72), suggesting that purulence in the stable state was associated with low grade pulmonary inflammation or epithelial damage or both. Abnormalities of air flow were found in 24 of the patients (70.6%) but there was a significantly higher ratio of residual volume to total lung capacity (p less than 0.025) in patients who regularly produced purulent sputum (mean (SD) RV/TLC 44.4% (9.0%] than in those with mucoid or mucopurulent secretions (38.0% (9.9%]. A similar difference was found between those who produced elastase positive secretions and those who produced elastase negative ones. PMID:6565423

Reproducibility of biomarkers in induced sputum and in serum from chronic smokers.

PubMed

Zuiker, Rob G J A; Kamerling, Ingrid M C; Morelli, Nicoletta; Calderon, Cesar; Boot, J Diderik; de Kam, Marieke; Diamant, Zuzana; Burggraaf, Jacobus; Cohen, Adam F

2015-08-01

Soluble inflammatory markers obtained from non-invasive airway sampling such as induced sputum may be useful biomarkers for targeted pharmaceutical interventions. However, before these soluble markers can be used as potential targets, their variability and reproducibility need to be established in distinct study populations. This study aimed to assess the reproducibility of biomarkers obtained from induced sputum and serum in chronic smokers and non-smokers. Sputum and serum samples were obtained from 16 healthy non-smokers and 16 asymptomatic chronic smokers (for both groups: 8M/8F, 30-52 years, FEV1 ≥80% pred.; ≥10 pack years for the smokers) on 2 separate visits 4-10 days apart. Soluble markers in serum and sputum were analysed by ELISA. The differences between smokers vs non-smokers were analysed with a t-test and variability was assessed on log-transformed data by a mixed model ANOVA. Analysable sputum samples could be obtained from all 32 subjects. In both study populations neutrophils and macrophages were the predominant cell types. Serum Pulmonary Surfactant Associated Protein D had favourable reproducibility criteria for reliability ratio (0.99), intra-subject coefficient of variation (11.2%) and the Bland Altman limits of agreement. Furthermore, chronic smokers, compared to non-smokers, had significantly higher sputum concentrations of IL-8 (1094.6 pg/mL vs 460.8 pg/mL, p = 0.006)), and higher serum concentrations of Pulmonary Surfactant Associated Protein D (110.9 pg/mL vs 64.7 pg/mL, p = 0.019), and lower concentrations of Serum Amyloid A (1352.4 pg/mL vs 2297.5 pg/mL, p = 0.022). Serum Pulmonary Surfactant Associated Protein D proved to be a biomarker that fulfilled the criteria for reproducibility in both study groups. Copyright © 2015 Elsevier Ltd. All rights reserved.

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

PubMed Central

Anthwal, Divya; Gupta, Rakesh Kumar; Bhalla, Manpreet; Bhatnagar, Shinjini

2017-01-01

ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INH-inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases. PMID:28330890

DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

EPA Science Inventory

Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
<...

The Diagnostic Accuracy of Urine-Based Xpert MTB/RIF in HIV-Infected Hospitalized Patients Who Are Smear-Negative or Sputum Scarce

PubMed Central

Peter, Jonathan G.; Theron, Grant; Muchinga, Tapuwa E.; Govender, Ureshnie; Dheda, Keertan

2012-01-01

Background Hospitals in sub-Saharan Africa are inundated with HIV-infected patients and tuberculosis (TB) is the commonest opportunistic infection in this sub-group. Up to one third of TB-HIV co-infected patients fail to produce a sputum sample (sputum scarce) and diagnosis is thus often delayed or missed. We investigated the sensitivity of urine-based methods (Xpert MTB/RIF, LAM strip test and LAM ELISA) in such patients. Methodology/Principal Findings 281 HIV-infected hospitalised patients with clinically suspected TB provided a spot urine sample. The reference standard was culture positivity for Mycobacterium tuberculosis on ≥1 sputum or extra-pulmonary sample. MTB/RIF was performed using 1 ml of both unprocessed and, when possible, concentrated urine. Each unconcentrated urine sample was also tested using the Clearview LAM ELISA and Alere LAM strip test. 42% (116/242) of patients had culture-proven TB. 18% (20/54) were sputum scarce. In sputum-scarce patients, the sensitivity of urine MTB/RIF and LAM ELISA was 40% (95%CI: 22–61) and 60% (95%CI: 39–78), respectively. Urine MTB/RIF specificity was 98% (95%CI: 95–100). Combined sensitivity of urine LAM ELISA and MTB/RIF was better than MTB/RIF alone [MTB/RIF and LAM: 70% (95%CI: 48–85) vs. MTB/RIF: 40% (95%CI: 22–61), p = 0.03]. Significant predictors of urine MTB/RIF positivity were CD4<50 cells/ml (p = 0.001), elevated protein-to-creatinine ratio (p<0.001) and LAM ELISA positivity (p<0.001). Urine centrifugation and pelleting significantly increased the sensitivity of MTB/RIF over unprocessed urine in paired samples [42% (95%CI: 26–58) vs. 8% (95%CI: 0–16), p<0.001]. Urine MTB/RIF-generated CT values correlated poorly with markers of bacillary burden (smear grade and time-to-positivity). Conclusions/Significance This preliminary study indicates that urine-based MTB/RIF, alone or in combination with LAM antigen detection, may potentially aid the diagnosis of TB in HIV-infected patients with advanced immunosuppression when sputum-based diagnosis is not possible. Concentration of urine prior to MTB/RIF-testing significantly improves sensitivity. PMID:22815718

Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.

PubMed

Moreau-Marquis, Sophie; Coutermarsh, Bonita; Stanton, Bruce A

2015-01-01

Chelating iron may be a promising new therapy to eliminate Pseudomonas aeruginosa biofilms in the lungs of cystic fibrosis (CF) patients. Here, we investigate whether ALX-109 [a defined combination of an investigational drug containing lactoferrin (an iron-binding glycoprotein) and hypothiocyanite (a bactericidal agent)], alone and in combination with tobramycin or aztreonam, reduces P. aeruginosa biofilms grown on human CF airway epithelial cells. P. aeruginosa (PAO1 and six clinical isolates of Pseudomonas) biofilms grown at the apical surface of confluent monolayers of CF airway epithelial cells were treated with ALX-109, either alone or in combination with tobramycin or aztreonam. Bacterial cfu remaining after treatment were determined by plate counting. ALX-109 alone reduced PAO1 biofilm formation, but had no effect on established biofilms. ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit PAO1 biofilm formation and to reduce established PAO1 biofilms. ALX-109 and tobramycin were additive in disrupting established biofilms formed by six clinical isolates of P. aeruginosa obtained from the sputum of CF patients. Mucoid P. aeruginosa isolates were most susceptible to the combination of ALX-109 and tobramycin. In addition, ALX-109 also enhanced the ability of aztreonam to reduce established PAO1 biofilms. Inhalation therapy combining hypothiocyanite and lactoferrin with TOBI(®) (tobramycin) or Cayston(®) (aztreonam) may be beneficial to CF patients by decreasing the airway bacterial burden of P. aeruginosa. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation.

PubMed

De Stefano, Daniela; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; De Gregorio, Fabiola; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; De Rosa, Giuseppe; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

2014-01-01

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Sputum induction is a safe procedure to use in prisoners and MGIT is the best culture method to diagnose tuberculosis in prisons: a cohort study.

PubMed

Rueda, Zulma Vanessa; López, Lucelly; Marín, Diana; Vélez, Lázaro A; Arbeláez, María Patricia

2015-04-01

To evaluate the concordance and safety of induced sputum (IS) and spontaneous sputum (SS), and estimate concordance and time to detection of M. tuberculosis between Lowenstein-Jensen (LJ), thin-layer agar (TLA), and the Mycobacteria Growth Indicator Tube system (MGIT). This was a cohort study. Prisoners with pulmonary tuberculosis (PTB) were followed for 2 years. At baseline and every follow-up visit, three sputum samples were taken on consecutive days (one IS and two SS) and adverse events occurring before, during, and 30 min after IS were registered. All sputum samples were stained with auramine and cultured in LJ, TLA (to test resistance), and MGIT. Five hundred eighty-six IS and 532 SS were performed on 64 PTB patients. Breathlessness (1.6%), cough (1.2%), hemoptysis (0.3%), and cyanosis (0.2%) were the only complications. Concordance between IS and SS was 0.78 (95% confidence interval 0.69-0.87); 11 positive cultures from IS samples were negative in SS, and 11 positive cultures from SS samples were negative in IS. One hundred seventy-eight cultures were positive by any technique: MGIT 95%, LJ 73%, and TLA 57%. Time to detection of M. tuberculosis in LJ, TLA, and MGIT was 31, 18, and 11 days, respectively. The IS procedure is safe in prisons. The MGIT system is better and faster than LJ and TLA in the diagnosis of M. tuberculosis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Efficiency of the Lung Flute for sputum induction in patients with presumed pulmonary tuberculosis.

PubMed

Sakashita, Kentaro; Fujita, Akira; Takamori, Mikio; Nagai, Takayuki; Matsumoto, Tomoshige; Saito, Takefumi; Nakagawa, Taku; Ogawa, Kenji; Shigeto, Eriko; Nakatsumi, Yasuto; Goto, Hajime; Mitarai, Satoshi

2018-04-01

High quality sputum helps increase the sensitivity of the diagnosis of pulmonary tuberculosis. To evaluate the efficiency of the acoustic device (Lung Flute; LF) in sputum induction compared with the conventional method, hypertonic saline inhalation (HSI). In this crossover study, patients with presumed pulmonary tuberculosis submitted 3 consecutive sputa: the first sputum without induction and the second and third ones using LF and HSI. We compared the efficiency of the 2 induction methods. Sixty-four participants were eligible. Thirty-five (54.6%) patients had negative smears on the first sputum without induction. Among those patients, 25.7% and 22.9% patients were smear-positive after using LF and HSI, respectively (P = .001). The positive conversion rate was not significantly different between the methods. The first samples without induction yielded 65.7% positive cultures, whereas 71.4% and 77.1% of the samples from LF and HSI were positive, respectively (P = .284). Similar results were observed in the nucleic acid amplification test [no induction (60.0%), LF (72.0%) and HSI (60.0%); P = .341]. In 29 smear-positive patients on the first sputum without induction, we observed no significant increase in smear grade, culture yield and nucleic acid amplification test positivity with either method. LF tended to induce fewer adverse events; desaturation (3.1% vs 11.1%; P = .082) and throat pain (1.5% vs 9.5%; P = .057). LF showed significantly fewer total adverse events (15.8% vs 34.9%; P = .023). Our study showed LF had similar sputum induction efficiency to HSI with relatively fewer complications. © 2017 The Authors. The Clinical Respiratory Journal Published by John Wiley & Sons Ltd.

Correlation of total serum immunoglobulin E level, sputum, and peripheral eosinophil count in assessing the clinical severity in bronchial asthma.

PubMed

Kumar, Roshan M; Pajanivel, R; Koteeswaran, G; Menon, Surendra K; Charles, Pravin Mv

2017-01-01

Asthma is a chronic inflammatory disorder of the airway with involvement of various cellular populations and release of many inflammatory mediators. Eosinophils and serum immunoglobulin E (IgE) are considered a good marker of airway inflammation in asthma. The correlation of clinical assessment with various markers of airway inflammation in asthma is not well established in the Indian population. This study aims to study the correlation of serum IgE, sputum eosinophil count, and peripheral eosinophil count with clinical severity of Asthma. This is a cross-sectional study involving 76 stable asthmatic patients of 18-60 years of age attending the pulmonary medicine OPD. Spirometry measured at baseline. Participants were categorized according to the GINA criteria based on clinical symptoms and pulmonary function test. Blood samples were collected for peripheral eosinophil count, serum IgE levels, and sputum samples for eosinophil count. All three parameters were compared with severity of asthma. The correlation of sputum eosinophil count, peripheral eosinophil count, and serum IgE with severity of asthma was analyzed by Pearson's Chi-square test, Fisher's exact test, and the correlation coefficient was reported together with standard error of the estimate. The mean age of patients in our study was 37.42 years and 56.6% were male. There was a significant inverse correlation between serum IgE levels and predicted forced expiratory volume 1 s (FEV1). Sputum eosinophilia was significantly seen in severe persistent asthma patients (19.7%). There was a significant inverse correlation between sputum eosinophil count and predicted FEV1and forced vital capacity. We also found there was a significant association between peripheral eosinophil count, sputum eosinophil count, and elevated serum IgE (g100 IU/mL) with severe persistent asthma. The assessment of sputum eosinophil count is simple, inexpensive, noninvasive, and direct measurement of airway inflammation. It could be the preferred method in monitoring airway inflammation and guided management in day-to-day practice.

Antibody Responses to Citrullinated and Noncitrullinated Antigens in the Sputum of Subjects With Rheumatoid Arthritis and Subjects at Risk for Development of Rheumatoid Arthritis.

PubMed

Demoruelle, M Kristen; Bowers, Emily; Lahey, Lauren J; Sokolove, Jeremy; Purmalek, Monica; Seto, Nickie L; Weisman, Michael H; Norris, Jill M; Kaplan, Mariana J; Holers, V Michael; Robinson, William H; Deane, Kevin D

2018-04-01

The location and mechanisms involved in the initial generation of autoantibodies to citrullinated and noncitrullinated proteins/peptides during the natural history of rheumatoid arthritis (RA) development is incompletely understood. This study sought to explore individual antibody responses to citrullinated and noncitrullinated proteins/peptides in the sputum and associations with neutrophil extracellular traps (NETs) in subjects at risk for the future development of RA. Serum and sputum samples were obtained from 41 RA-free subjects who were considered at risk for the development of RA based on familial or serologic risk factors, from 20 subjects classified as having RA, and from 22 healthy control subjects. Samples were evaluated using a bead-based array for IgG reactivity to 29 citrullinated proteins/peptides and 21 noncitrullinated proteins/peptides. Cutoff levels for antibody positivity were established in a separate control group. NET levels in the sputum were measured using sandwich enzyme-linked immunosorbent assays that quantitate DNA-myeloperoxidase and DNA-neutrophil elastase complexes. In at-risk subjects, antibody responses to the citrullinated forms of fibrinogen, apolipoprotein E, and fibronectin were highly prevalent. The most citrulline-specific antibodies in the sputum of at-risk subjects were those to fibrinogen, vimentin, and peptides of fibrinogen A and apolipoprotein A1. Patterns of sputum autoantibody positivity differed between at-risk subjects and subjects with RA. In at-risk subjects, increasing sputum NET levels significantly correlated with several citrullinated and some noncitrullinated antibody reactivities. These findings suggest that sputum antibody reactivity to particular citrullinated and noncitrullinated proteins/peptides is specific for RA and for subjects at risk of RA, and the association of these proteins/peptides with NETs may be a key feature of early RA-related autoimmunity in the lung. These results further support the hypothesis that the lung plays a role in early RA-related autoimmunity. © 2017, American College of Rheumatology.

Novel multi-day sputum transport reagent works with routine tuberculosis tests and eliminates need for cold chain: Preliminary study of compatibility with the Xpert® MTB/RIF assay.

PubMed

Kelly-Cirino, Cassandra D; Curry, Patricia S; Marola, Jamie L; Helstrom, Niels K; Salfinger, Max

2016-11-01

OMNIgene®•SPUTUM (OM-S) is a sputum transport reagent designed to work with all tuberculosis diagnostics and eliminate the need for cold chain. The aim of this preliminary study was to assess the compatibility of OM-S-treated sputum with the Xpert® MTB/RIF assay. Fifty-five characterized sputa from the FIND TB Specimen Bank were used. Compatibility of OM-S was assessed for both Xpert sample preparation methods: H.1 protocol (sediment, n=25) and H.2 protocol (direct expectorate, n=30). All controls were prepared using the H.2 protocol. Results revealed 100% concordance of MTB/RIF results for all except the low-positive group in the H.1 study arm (n=10; 88% concordance). OM-S-treated sputa were successful in both protocols; if the Xpert buffer is not added during the H.2 procedure, sample viscosity may require repeat testing. Using OM-S could offer users flexibility in clinical testing algorithms. Larger compatibility studies are warranted, particularly with respect to MTB/RIF results for low-positive samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of Candida species isolated from cases of lower respiratory tract infection.

PubMed

Jha, B J; Dey, S; Tamang, M D; Joshy, M E; Shivananda, P G; Brahmadatan, K N

2006-01-01

(1) To identify and characterize the Candida species isolates from lower respiratory tract infection. (2) to determine the rate of isolation of Candida species from sputum samples. This study was carried out in the Department of Microbiology, Manipal Teaching Hospital, Pokhara, Nepal from June 2002 to January 2003. A total of 462 sputum samples were collected from patients suspected lower respiratory tract infection. The samples were processed as Gram staining to find out the suitability of the specimen, cultured on Sabouraud's Dextrose Agar (SDA) and also on blood agar and chocolate agar to identify the potential lower respiratory tract pathogens. For the identification of Candida, sputum samples were processed for Gram stain, culture, germ tube test, production of chlamydospore, sugar fermentation and assimilation test. For the identification of bacteria, Gram stain, culture, and biochemical tests were performed by standardized procedure. Out of 462 samples, 246 (53.24%) samples grew potential pathogens of lower respiratory tract. Among them Haemophilus influenzae 61(24.79%) and Streptococcus pneumoniae 57 (23.17%) were the predominant bacterial pathogens. Candida species were isolated from 30 samples (12.2%). The majority of Candida species amongst the Candida isolates were Candida albicans 21(70%) followed by Candida tropicalis 4(13.33%). Candida krusei 3(10%), Candida parapsilosis 1(3.33%) and Candida stellatoidea 1(3.33%). The highest rate of isolation of Candida was between the age of 71 and 80. Candida isolation from sputum samples is important as found in the present study in which Candida species were the third most common pathogen isolated from patients with lower respiratory tract infection.

Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung.

PubMed

Naughton, Sharna; Parker, Dane; Seemann, Torsten; Thomas, Torsten; Turnbull, Lynne; Rose, Barbara; Bye, Peter; Cordwell, Stuart; Whitchurch, Cynthia; Manos, Jim

2011-01-01

Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.

Pseudomonas aeruginosa AES-1 Exhibits Increased Virulence Gene Expression during Chronic Infection of Cystic Fibrosis Lung

PubMed Central

Naughton, Sharna; Parker, Dane; Seemann, Torsten; Thomas, Torsten; Turnbull, Lynne; Rose, Barbara; Bye, Peter; Cordwell, Stuart; Whitchurch, Cynthia; Manos, Jim

2011-01-01

Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients. PMID:21935417

The Potential Role of Lung Microbiota in Lung Cancer Attributed to Household Coal Burning Exposures

PubMed Central

Hosgood, H. Dean; Sapkota, Amy R.; Rothman, Nathaniel; Rohan, Thomas; Hu, Wei; Xu, Jun; Vermeulen, Roel; He, Xingzhou; White, James Robert; Wu, Guoping; Wei, Fusheng; Mongodin, Emmanuel F.; Lan, Qing

2014-01-01

Bacteria influence site-specific disease etiology and the host’s ability to metabolize xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs). Lung cancer in Xuanwei, China has been attributed to PAH-rich household air pollution from burning coal. This study seeks to explore the role of lung microbiota in lung cancer among never smoking Xuanwei women and how coal burning may influence these associations. DNA from sputum and buccal samples of never smoking lung cancer cases (n = 8, in duplicate) and controls (n = 8, in duplicate) in two Xuanwei villages was extracted using a multi-step enzymatic and physical lysis, followed by a standardized clean-up. V1–V2 regions of 16S rRNA genes were PCR-amplified. Purified amplicons were sequenced by 454 FLX Titanium pyrosequencing and high-quality sequences were evaluated for diversity and taxonomic membership. Bacterial diversity among cases and controls was similar in buccal samples (P = 0.46), but significantly different in sputum samples (P = 0.038). In sputum, Granulicatella (6.1 vs. 2.0%; P = 0.0016), Abiotrophia (1.5 vs. 0.085%; P = 0.0036), and Streptococcus (40.1 vs. 19.8%; P = 0.0142) were enriched in cases compared with controls. Sputum samples had on average 488.25 species-level OTUs in the flora of cases who used smoky coal (PAHrich) compared with 352.5 OTUs among cases who used smokeless coal (PAH-poor; P = 0.047). These differences were explained by the Bacilli species (Streptococcus infantis and Streptococcus anginosus). Our small study suggests that never smoking lung cancer cases have differing sputum microbiota than controls. PMID:24895247

Destruction-free procedure for the isolation of bacteria from sputum samples for Raman spectroscopic analysis.

PubMed

Kloß, Sandra; Lorenz, Björn; Dees, Stefan; Labugger, Ines; Rösch, Petra; Popp, Jürgen

2015-11-01

Lower respiratory tract infections are the fourth leading cause of death worldwide. Here, a timely identification of the causing pathogens is crucial to the success of the treatment. Raman spectroscopy allows for quick identification of bacterial cells without the need for time-consuming cultivation steps, which is the current gold standard to detect pathogens. However, before Raman spectroscopy can be used to identify pathogens, they have to be isolated from the sample matrix, i.e., sputum in case of lower respiratory tract infections. In this study, we report an isolation protocol for single bacterial cells from sputum samples for Raman spectroscopic identification. Prior to the isolation, a liquefaction step using the proteolytic enzyme mixture Pronase E is required in order to deal with the high viscosity of sputum. The extraction of the bacteria was subsequently performed via different filtration and centrifugation steps, whereby isolation ratios between 46 and 57 % were achieved for sputa spiked with 6·10(7) to 6·10(4) CFU/mL of Staphylococcus aureus. The compatibility of such a liquefaction and isolation procedure towards a Raman spectroscopic classification was shown for five different model species, namely S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. A classification of single-cell Raman spectra of these five species with an accuracy of 98.5 % could be achieved on the basis of a principal component analysis (PCA) followed by a linear discriminant analysis (LDA). These classification results could be validated with an independent test dataset, where 97.4 % of all spectra were identified correctly. Graphical Abstract Development of an isolation protocol of bacterial cells out of sputum samples followed by Raman spectroscopic measurement and species identification using chemometrical models.

Proteomics of Pseudomonas aeruginosa Australian epidemic strain 1 (AES-1) cultured under conditions mimicking the cystic fibrosis lung reveals increased iron acquisition via the siderophore pyochelin.

PubMed

Hare, Nathan J; Soe, Cho Zin; Rose, Barbara; Harbour, Colin; Codd, Rachel; Manos, Jim; Cordwell, Stuart J

2012-02-03

Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R.

Direct microscopy versus sputum cytology analysis and bleach sedimentation for diagnosis of tuberculosis: a prospective diagnostic study

PubMed Central

2010-01-01

Background Diagnostic options for pulmonary tuberculosis in resource-poor settings are commonly limited to smear microscopy. We investigated whether bleach concentration by sedimentation and sputum cytology analysis (SCA) increased the positivity rate of smear microscopy for smear-positive tuberculosis. Methods We did a prospective diagnostic study in a Médecins Sans Frontières-supported hospital in Mindouli, Republic of Congo. Three sputum samples were obtained from 280 consecutive pulmonary tuberculosis suspects, and were processed according to WHO guidelines for direct smear microscopy. The remainder of each sputum sample was homogenised with 2.6% bleach, sedimented overnight, smeared, and examined blinded to the direct smear result for acid-fast bacilli (AFB). All direct smears were assessed for quality by SCA. If a patient produced fewer than three good-quality sputum samples, further samples were requested. Sediment smear examination was performed independently of SCA result on the corresponding direct smear. Positivity rates were compared using McNemar's test. Results Excluding SCA, 43.2% of all patients were diagnosed as positive on direct microscopy of up to three samples. 47.9% were diagnosed on sediment microscopy, with 48.2% being diagnosed on direct microscopy, sediment microscopy, or both. The positivity rate increased from 43.2% to 47.9% with a case definition of one positive smear (≥1 AFB/100 high power fields) of three, and from 42.1% to 43.9% with two positive smears. SCA resulted in 87.9% of patients producing at least two good-quality sputum samples, with 75.7% producing three or more. Using a case definition of one positive smear, the incremental yield of bleach sedimentation was 14/121, or 11.6% (95% CI 6.5-18.6, p = 0.001) and in combination with SCA was 15/121, or 12.4% (95% CI 7.1-19.6, p = 0.002). Incremental yields with two positive smears were 5/118, or 4.2% (95% CI 1.4-9.6, p = 0.062) and 7/118, or 5.9% (95% CI 2.4-11.8, p = 0.016), respectively. Conclusions The combination of bleach sedimentation and SCA resulted in significantly increased microscopy positivity rates with a case definition of either one or two positive smears. Implementation of bleach sedimentation led to a significant increase in the diagnosis of smear-positive patients. Implementation of SCA did not result in significantly increased diagnosis of tuberculosis, but did result in improved sample quality. Requesting extra sputum samples based on SCA results, combined with bleach sedimentation, could significantly increase the detection of smear-positive patients if routinely implemented in resource-limited settings where gold standard techniques are not available. We recommend that a pilot phase is undertaken before routine implementation to determine the impact in a particular context. PMID:20858253

A Biophysical Basis for Mucus Solids Concentration as a Candidate Biomarker for Airways Disease

PubMed Central

Hill, David B.; Vasquez, Paula A.; Mellnik, John; McKinley, Scott A.; Vose, Aaron; Mu, Frank; Henderson, Ashley G.; Donaldson, Scott H.; Alexis, Neil E.; Boucher, Richard C.; Forest, M. Gregory

2014-01-01

In human airways diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), host defense is compromised and airways inflammation and infection often result. Mucus clearance and trapping of inhaled pathogens constitute key elements of host defense. Clearance rates are governed by mucus viscous and elastic moduli at physiological driving frequencies, whereas transport of trapped pathogens in mucus layers is governed by diffusivity. There is a clear need for simple and effective clinical biomarkers of airways disease that correlate with these properties. We tested the hypothesis that mucus solids concentration, indexed as weight percent solids (wt%), is such a biomarker. Passive microbead rheology was employed to determine both diffusive and viscoelastic properties of mucus harvested from human bronchial epithelial (HBE) cultures. Guided by sputum from healthy (1.5–2.5 wt%) and diseased (COPD, CF; 5 wt%) subjects, mucus samples were generated in vitro to mimic in vivo physiology, including intermediate range wt% to represent disease progression. Analyses of microbead datasets showed mucus diffusive properties and viscoelastic moduli scale robustly with wt%. Importantly, prominent changes in both biophysical properties arose at ∼4 wt%, consistent with a gel transition (from a more viscous-dominated solution to a more elastic-dominated gel). These findings have significant implications for: (1) penetration of cilia into the mucus layer and effectiveness of mucus transport; and (2) diffusion vs. immobilization of micro-scale particles relevant to mucus barrier properties. These data provide compelling evidence for mucus solids concentration as a baseline clinical biomarker of mucus barrier and clearance functions. PMID:24558372

Successful recovery after disseminated infection due to mycobacterium abscessus in a lung transplant patient: subcutaneous nodule as first manifestation--a case report.

PubMed

Morales, P; Ros, J A; Blanes, M; Pérez-Enguix, D; Saiz, V; Santos, M

2007-09-01

Mycobacterium abscessus infection following lung transplantation (LT) has been described in a few cases. It is characterized by a variable initial location and subsequent course in this special risk group of patients, particularly those with cystic fibrosis (CF). Herein we have presented the case of a patient subjected to LT due to CF, who 1 year after transplantation developed a subcutaneous nodule produced by M abscessus, with subsequent hematogenous spread as well as bronchial and bone marrow involvement. Antecedents prior to LT included Staphylococcus aureus colonization and sputum positivity for Aspergillus fumigatus and Scedosporium apioespermum. Treatment with ciprofloxacin and linezolid was started on the basis of the antibiogram findings. The latter antibiotic was replaced by clarithromycin for 6 months. Two years later, the patient remains asymptomatic with respiratory function parameters in the normal range. The infected patient described herein was our only case with sepsis and multisystemic spread. The important mortality among such cases reported in the literature makes early diagnosis and treatment essential.

A novel automatic molecular test for detection of multidrug resistance tuberculosis in sputum specimen: A case control study.

PubMed

Li, Qiang; Ou, Xi C; Pang, Yu; Xia, Hui; Huang, Hai R; Zhao, Bing; Wang, Sheng F; Zhao, Yan L

2017-07-01

MiniLab tuberculosis (ML TB) assay is a new automatic diagnostic tool for diagnosis of multidrug resistance tuberculosis (MDR-TB). This study was conducted with aims to know the performance of this assay. Sputum sample from 224 TB suspects was collected from tuberculosis suspects seeking medical care at Beijing Chest hospital. The sputum samples were directly used for smear and ML TB test. The left sputum sample was used to conduct Xpert MTB/RIF, Bactec MGIT culture and drug susceptibility test (DST). All discrepancies between the results from DST, molecular and phenotypic methods were confirmed by DNA Sequencing. The sensitivity and specificity of ML TB test for detecting MTBC from TB suspects were 95.1% and 88.9%, respectively. The sensitivity for smear negative TB suspects was 64.3%. For detection of RIF resistance, the sensitivity and specificity of ML TB test were 89.2% and 95.7%, respectively. For detection of INH resistance, the sensitivity and specificity of ML TB test were 78.3% and 98.1%, respectively. ML TB test showed similar performance to Xpert MTB/RIF for detection of MTBC and RIF resistance. In addition, ML TB also had good performance for INH resistance detection. Copyright © 2017. Published by Elsevier Ltd.

A cross-sectional study of sputum handling by and supervision of patients with pulmonary tuberculosis treated at home in China.

PubMed

Mei, L; Tobe, R G; Geng, H; Ma, Y B; Li, R Y; Wang, W B; Selotlegeng, L; Wang, X Z; Xu, L Z

2012-12-01

Disposal of sputum from patients with pulmonary tuberculosis (TB) who are treated at home is an important aspect of preventing the spread of TB. However, few studies have examined disposal of sputum by patients with TB who are treated at home. Patients with pulmonary TB who are treated at home were surveyed regarding sputum handling and supervision. A cross-sectional survey of a representative sample of patients with pulmonary TB who are treated at home was conducted in Shandong Province. Participants were individuals with TB who had been registered with a local agency responsible for TB control. Participants completed a questionnaire with both qualitative and quantitative questions. How sputum was handled was determined and factors associated with sputum disposal were analyzed using a non-parametric test, logistic regression, and content analysis. Responses were received from 720 participants. Patients expectorated sputum 4.56 ± 10.367 times a day, and 68.6% of patients responded that they correctly disposed of their sputum. Supervision as part of TB control focused on the efforts of health agencies and paid little attention to waste management by patients. A non-parametric test showed that sputum disposal was significantly associated with gender, age, education, sputum smear results, attitudes toward waste management, and attitudes toward supervision (all p < 0.05). Logistic regression analysis showed that gender (OR = 0.482, 95% CI: 0.329-0.704), sputum smear results (OR = 1.300, 95% CI: 1.037-1.629), and level of education (OR = 0.685, 95% CI: 0.528-0.889) were associated with receipt of TB health education (all p < 0.05). Sputum handling by and supervision of patients with pulmonary TB who are treated at home is severely wanting. From a policy perspective, special attention should be given to the definition, details, and methods of supervision of waste management by patients with TB to give them relevant health education and enhance their willingness to be supervised. A financial incentive should be provided to health workers supervising management of TB-related waste.

Spot sputum samples are at least as good as early morning samples for identifying Mycobacterium tuberculosis.

PubMed

Murphy, Michael E; Phillips, Patrick P J; Mendel, Carl M; Bongard, Emily; Bateson, Anna L C; Hunt, Robert; Murthy, Saraswathi; Singh, Kasha P; Brown, Michael; Crook, Angela M; Nunn, Andrew J; Meredith, Sarah K; Lipman, Marc; McHugh, Timothy D; Gillespie, Stephen H

2017-10-27

The use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatment delay given the need for the patient to return to the clinic with the EMS, increasing the chance of patients being lost during their diagnostic workup. However, there is little evidence to support the superiority of EMS over spot sputum samples. In this new analysis of the REMoxTB study, we compare the diagnostic accuracy of EMS with spot samples for identifying Mycobacterium tuberculosis pre- and post-treatment. Patients who were smear positive at screening were enrolled into the study. Paired sputum samples (one EMS and one spot) were collected at each trial visit pre- and post-treatment. Microscopy and culture on solid LJ and liquid MGIT media were performed on all samples; those missing corresponding paired results were excluded from the analyses. Data from 1115 pre- and 2995 post-treatment paired samples from 1931 patients enrolled in the REMoxTB study were analysed. Patients were recruited from South Africa (47%), East Africa (21%), India (20%), Asia (11%), and North America (1%); 70% were male, median age 31 years (IQR 24-41), 139 (7%) co-infected with HIV with a median CD4 cell count of 399 cells/μL (IQR 318-535). Pre-treatment spot samples had a higher yield of positive Ziehl-Neelsen smears (98% vs. 97%, P = 0.02) and LJ cultures (87% vs. 82%, P = 0.006) than EMS, but there was no difference for positivity by MGIT (93% vs. 95%, P = 0.18). Contaminated and false-positive MGIT were found more often with EMS rather than spot samples. Surprisingly, pre-treatment EMS had a higher smear grading and shorter time-to-positivity, by 1 day, than spot samples in MGIT culture (4.5 vs. 5.5 days, P < 0.001). There were no differences in time to positivity in pre-treatment LJ culture, or in post-treatment MGIT or LJ cultures. Comparing EMS and spot samples in those with unfavourable outcomes, there were no differences in smear or culture results, and positive results were not detected earlier in Kaplan-Meier analyses in either EMS or spot samples. Our data do not support the hypothesis that EMS samples are superior to spot sputum samples in a clinical trial of patients with smear positive pulmonary TB. Observed small differences in mycobacterial burden are of uncertain significance and EMS samples do not detect post-treatment positives any sooner than spot samples.

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation.

PubMed

Tong, Yu; Shen, Shizhen; Jiang, Hui; Chen, Zhi

2017-01-01

Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations. © 2017 The Author(s). Published by S. Karger AG, Basel.

Convergent Metabolic Specialization through Distinct Evolutionary Paths in Pseudomonas aeruginosa

PubMed Central

Johansen, Helle Krogh; Molin, Søren

2018-01-01

ABSTRACT Evolution by natural selection under complex and dynamic environmental conditions occurs through intricate and often counterintuitive trajectories affecting many genes and metabolic solutions. To study short- and long-term evolution of bacteria in vivo, we used the natural model system of cystic fibrosis (CF) infection. In this work, we investigated how and through which trajectories evolution of Pseudomonas aeruginosa occurs when migrating from the environment to the airways of CF patients, and specifically, we determined reduction of growth rate and metabolic specialization as signatures of adaptive evolution. We show that central metabolic pathways of three distinct Pseudomonas aeruginosa lineages coevolving within the same environment become restructured at the cost of versatility during long-term colonization. Cell physiology changes from naive to adapted phenotypes resulted in (i) alteration of growth potential that particularly converged to a slow-growth phenotype, (ii) alteration of nutritional requirements due to auxotrophy, (iii) tailored preference for carbon source assimilation from CF sputum, (iv) reduced arginine and pyruvate fermentation processes, and (v) increased oxygen requirements. Interestingly, although convergence was evidenced at the phenotypic level of metabolic specialization, comparative genomics disclosed diverse mutational patterns underlying the different evolutionary trajectories. Therefore, distinct combinations of genetic and regulatory changes converge to common metabolic adaptive trajectories leading to within-host metabolic specialization. This study gives new insight into bacterial metabolic evolution during long-term colonization of a new environmental niche. PMID:29636437

Do Instructional Videos on Sputum Submission Result in Increased Tuberculosis Case Detection? A Randomized Controlled Trial.

PubMed

Mhalu, Grace; Hella, Jerry; Doulla, Basra; Mhimbira, Francis; Mtutu, Hawa; Hiza, Helen; Sasamalo, Mohamed; Rutaihwa, Liliana; Rieder, Hans L; Seimon, Tamsyn; Mutayoba, Beatrice; Weiss, Mitchell G; Fenner, Lukas

2015-01-01

We examined the effect of an instructional video about the production of diagnostic sputum on case detection of tuberculosis (TB), and evaluated the acceptance of the video. Randomized controlled trial. We prepared a culturally adapted instructional video for sputum submission. We analyzed 200 presumptive TB cases coughing for more than two weeks who attended the outpatient department of the governmental Municipal Hospital in Mwananyamala (Dar es Salaam, Tanzania). They were randomly assigned to either receive instructions on sputum submission using the video before submission (intervention group, n = 100) or standard of care (control group, n = 100). Sputum samples were examined for volume, quality and presence of acid-fast bacilli by experienced laboratory technicians blinded to study groups. Median age was 39.1 years (interquartile range 37.0-50.0); 94 (47%) were females, 106 (53%) were males, and 49 (24.5%) were HIV-infected. We found that the instructional video intervention was associated with detection of a higher proportion of microscopically confirmed cases (56%, 95% confidence interval [95% CI] 45.7-65.9%, sputum smear positive patients in the intervention group versus 23%, 95% CI 15.2-32.5%, in the control group, p <0.0001), an increase in volume of specimen defined as a volume ≥3ml (78%, 95% CI 68.6-85.7%, versus 45%, 95% CI 35.0-55.3%, p <0.0001), and specimens less likely to be salivary (14%, 95% CI 7.9-22.4%, versus 39%, 95% CI 29.4-49.3%, p = 0.0001). Older age, but not the HIV status or sex, modified the effectiveness of the intervention by improving it positively. When asked how well the video instructions were understood, the majority of patients in the intervention group reported to have understood the video instructions well (97%). Most of the patients thought the video would be useful in the cultural setting of Tanzania (92%). Sputum submission instructional videos increased the yield of tuberculosis cases through better quality of sputum samples. If confirmed in larger studies, instructional videos may have a substantial effect on the case yield using sputum microscopy and also molecular tests. This low-cost strategy should be considered as part of the efforts to control TB in resource-limited settings. Pan African Clinical Trials Registry PACTR201504001098231.

Investigating the quality of expectorated sputum for tuberculosis diagnosis in Bolivia.

PubMed

Hernández, P; Punchak, M; Camacho, M; Hepple, P; McNerney, R

2015-09-01

A low-power microscope-based cytological system to assess the quality of expectorated sputum provided for tuberculosis (TB) diagnosis was piloted in Bolivia. A total of 3688 samples were subjected to visual and cytological examination in nine laboratories: of these, 591 (16%) were misclassified by visual examination and 294 (8%) were found to be degraded. The degree of discordance varied between locations, and laboratories received a higher number of degraded specimens from isolated health clinics. Cytological assessment of sputum was found to be feasible and identified areas for improvement in the Bolivian diagnostic system for TB.

Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways

PubMed Central

Lay, John C.; Peden, David B.; Alexis, Neil E.

2012-01-01

Background The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. Objective To develop a gating strategy based on specific antibody panels in combination with light scatter properties for flow cytometric evaluation of sputum cells. Methods Healthy and mild asthmatic volunteers underwent sputum induction. Manually selected mucus “plug” material was treated with dithiothrietol, filtered and total leukocytes acquired. Multicolor flow cytometry was performed using specific gating strategies based on light scatter properties, differential expression of CD45 and cell lineage markers to discriminate leukocytes from squamous epithelial cells and debris. Results The combination of forward scatter and CD45 expression reliably segregated sputum leukocytes from contaminating squamous epithelial cells and debris. Overlap of major leukocyte populations (neutrophils, macrophages/monocytes) required the use of specific antibodies (e.g. CD16, CD64, CD14, HLA-DR) that differentiated granulocytes from monocytes and macrophages. These gating strategies allowed identification of small populations of eosinophils, CD11c+ myeloid dendritic cells, B cells and NK cells. Conclusions Multicolor flow cytometry can be successfully applied to sputum samples to identify and characterize leukocyte populations residing on the surfaces of the central airways. PMID:21639708

Infectious hematopoietic necrosis virus detected by separation and incubation of cells from salmonid cavity fluid.

USGS Publications Warehouse

Mulcahy, D.; Batts, W.N.

1987-01-01

Infectious hematopoietic necrosis (IHN) virus is usually detected by inoculating susceptible cell cultures with cavity ("ovarian") fluid (CF) from spawning females. We identified additional adult carriers of virus in spawning populations of steelhead trout (Salmo gairdneri) and sockeye salmon (Oncorhynchus nerka) by collecting nonerythrocytic cells from CF samples by low-speed centrifugation, culturing the cells for at least 7 d at 15 °C, and then testing the culture medium for virus. Virus appeared in the cultured cells from some samples of CF that remained negative during incubation. In additional samples of CF from these species, the virus titer increased in cultured cells compared with the titer in the original CF sample. With chinook salmon (O.tshawytscha), no negative samples converted to positive during incubation, but the virus titer was retained in incubated CF cells, but not in cell-free CF.

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS).

PubMed

Freeman, Christine M; Crudgington, Sean; Stolberg, Valerie R; Brown, Jeanette P; Sonstein, Joanne; Alexis, Neil E; Doerschuk, Claire M; Basta, Patricia V; Carretta, Elizabeth E; Couper, David J; Hastie, Annette T; Kaner, Robert J; O'Neal, Wanda K; Paine, Robert; Rennard, Stephen I; Shimbo, Daichi; Woodruff, Prescott G; Zeidler, Michelle; Curtis, Jeffrey L

2015-01-27

Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a "just-in-time" design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. This study was registered with ClinicalTrials.gov as NCT01969344 .

Evaluation of the GeneXpert MTB/RIF assay on extrapulmonary and respiratory samples other than sputum: a low burden country experience.

PubMed

Pandey, Sushil; Congdon, Jacob; McInnes, Bradley; Pop, Alina; Coulter, Christopher

2017-01-01

The aim of this study was to assess the performance of the GeneXpert MTB/RIF assay on extrapulmonary (EP) and respiratory (non-sputum) clinical samples of patients suspected of having tuberculosis (TB) from Queensland, Australia. A total of 269 EP and respiratory (non-sputum) clinical samples collected from Qld patients who were suspected of having TB were subjected to the GeneXpert MTB/RIF analysis, Ziehl-Neelsen (ZN) staining, Mycobacterium tuberculosis (MTB) culture and drug susceptibility testing. Phenotypic and genotypic data were compared. The overall performance analysis of the GeneXpert MTB/RIF assay for detection of MTB complex demonstrated sensitivity of 89%, specificity of 95%, PPV of 89% and NPV of 95% using culture as a reference standard. The GeneXpert MTB/RIF analysis of acid-fast bacilli (AFB) smear positive samples and AFB smear negative samples showed sensitivities of 100% and 77%, respectively. Looking at individual EP and respiratory (non-sputum) sample types, the sensitivity ranged from 60% to 100% although the specificity ranged from 33% to 100% with the specificity of lymph node tissue biopsy being the lowest. The GeneXpert MTB/RIF assay detected 11% more TB cases than culture and 27% more cases than ZN microscopy. Due to insufficient numbers of presenting rifampicin resistance cases, performance analysis of the GeneXpert MTB/RIF assay on rifampicin resistance could not be carried out. The GeneXpert MTB/RIF assay is potentially valuable for TB diagnosis in the majority of the EP and respiratory (other than sputum) samples in our setting. Although the GeneXpert MTB/RIF assay provides rapid diagnostic results, the overall sensitivity to rule out the disease is suboptimal for some specimen types. Performance varied according to specimen type and AFB smear status. The sensitivity and specificity of lymph node tissue was 63% and 33%. Care must be taken when using the GeneXpert MTB/RIF assay for detection of MTB in lymph node tissue samples. All samples should be cultured regardless of the GeneXpert MTB/RIF assay result. Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Protein Microarray Analysis in Patients With Asthma*

PubMed Central

Kim, Hyo-Bin; Kim, Chang-Keun; Iijima, Koji; Kobayashi, Takao; Kita, Hirohito

2010-01-01

Background Microarray technology offers a new opportunity to gain insight into global gene and protein expression profiles in asthma. To identify novel factors produced in the asthmatic airway, we analyzed sputum samples by using a membrane-based human cytokine microarray technology in patients with bronchial asthma (BA). Methods Induced sputum was obtained from 28 BA subjects, 20 nonasthmatic atopic control (AC) subjects, and 38 nonasthmatic nonatopic normal control (NC) subjects. The microarray samples of subjects were randomly selected from nine BA subjects, three AC subjects, and six NC subjects. Sputum supernatants were analyzed using a custom human cytokine array (RayBio Custom Human Cytokine Array; RayBiotech; Norcross, GA) designed to analyze 79 specific cytokines simultaneously. The levels of growth-regulated oncogene (GRO)-α, eotaxin-2, and pulmonary and activation-regulated chemokine (PARC)/CCL18 were measured by sandwich enzyme-linked immunosorbent assays (ELISAs), and eosinophil-derived neurotoxin (EDN) was measured by radioimmunoassay. Results By microarray, the signal intensities for GRO-α, eotaxin-2, and PARC were significantly higher in BA subjects than in AC and NC subjects (p = 0.036, p = 0.042, and p = 0.033, respectively). By ELISA, the sputum PARC protein levels were significantly higher in BA subjects than in AC and NC subjects (p < 0.0001). Furthermore, PARC levels correlated significantly with sputum eosinophil percentages (r = 0.570, p < 0.0001) and the levels of EDN(r = 0.633, p < 0.0001), the regulated upon activation, normal T cell expressed and secreted cytokine (r = 0.440, p < 0.001), interleukin-4 (r = 0.415, p < 0.01), and interferon-γ (r = 0.491, p < 0.001). Conclusions By a nonbiased screening approach, a chemokine, PARC, is elevated in sputum specimens from patients with asthma. PARC may play important roles in development of airway eosinophilic inflammation in asthma. PMID:19017877

A Halotyrosine Antibody that Detects Increased Protein Modifications in Asthma Patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Hongjun; Hallstrand, Teal S.; Daly, Don S.

Background-Airway inflammation plays an important pathophysiological role in asthma. Eosinophils produce hypobromite and bromotyrosine while neutrophils produce hypochlorite and chlorotyrosine. Objective-To evaluate halotyrosine modifications of individual airway proteins as a marker of inflammation in asthma using an antibody-based assay. Methods-We developed a novel monoclonal antibody (BTK-94C) that binds halogenated tyrosine residues, and used this antibody in a custom enzyme-linked immunosorbent assay (ELISA) microarray platform to examine halotyrosine levels in 23 proteins in three independent sets of sputum samples (52 samples total). Results-In 15 subjects with either no asthma, or with asthma characterized by high or low sputum eosinophil counts, wemore » found associations between increased halotyrosine levels of at least three proteins and severity of airway hyperresponsiveness (AHR). Treatment with mepolizumab in 17 patients with sputum eosinophilia markedly reduced the sputum eosinophilia and significantly reduced halotyrosine levels in one sputum protein. Further analysis of 10 subjects with neutrophilic asthma and 10 health controls demonstrated a broad increase in halotyrosine in the patients with airway neutrophilia. Conclusions-Significantly higher levels of halotyrosine are associated with asthma in the asthma phenotypes we examined. The halotyrosine levels correlated with indirect AHR in the form of exercise-induced bronchoconstriction. Clinical Implication-An antibody-based assay for tyrosine halogenation in specific proteins may prove useful for assessing airway inflammation in asthma. Capsule Summary-An antibody to measure protein monobrominated tyrosine and other halotyrosine modifications was developed and used to evaluate halogenation in specific proteins in the airways for the first time. Associations were found between levels of halotyrosine and exercise-induced bronchoconstriction, and eosinophil and neutrophil inflammation in sputum from asthma patients. The ELISA developed here can be easily translated into a clinical test.« less

Evaluation of efficiency of nested multiplex allele-specific PCR assay for detection of multidrug resistant tuberculosis directly from sputum samples.

PubMed

Mistri, S K; Sultana, M; Kamal, S M M; Alam, M M; Irin, F; Nessa, J; Ahsan, C R; Yasmin, M

2016-05-01

For an effective control of tuberculosis, rapid detection of multidrug resistant tuberculosis (MDR-TB) is necessary. Therefore, we developed a modified nested multiplex allele-specific polymerase chain reaction (MAS-PCR) method that enables rapid MDR-TB detection directly from sputum samples. The efficacy of this method was evaluated using 79 sputum samples collected from suspected tuberculosis patients. The performance of nested MAS-PCR method was compared with other MDR-TB detection methods like drug susceptibility testing (DST) and DNA sequencing. As rifampicin (RIF) resistance conforms to MDR-TB in greater than 90% cases, only the presence of RIF-associated mutations in rpoB gene was determined by DNA sequencing and nested MAS-PCR to detect MDR-TB. The concordance between nested MAS-PCR and DNA sequencing results was found to be 96·3%. When compared with DST, the sensitivity and specificity of nested MAS-PCR for RIF-resistance detection were determined to be 92·9 and 100% respectively. For developing- and high-TB burden countries, molecular-based tests have been recommended by the World Health Organization for rapid detection of MDR-TB. The results of this study indicate that, nested MAS-PCR assay might be a practical and relatively cost effective molecular method for rapid detection of MDR-TB from suspected sputum samples in developing countries with resource poor settings. © 2016 The Society for Applied Microbiology.

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

PubMed

Blakemore, Robert; Nabeta, Pamela; Davidow, Amy L; Vadwai, Viral; Tahirli, Rasim; Munsamy, Vanisha; Nicol, Mark; Jones, Martin; Persing, David H; Hillemann, Doris; Ruesch-Gerdes, Sabine; Leisegang, Felicity; Zamudio, Carlos; Rodrigues, Camilla; Boehme, Catharina C; Perkins, Mark D; Alland, David

2011-11-01

The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

Diagnostic dilemma of pulmonary tuberculosis among adults with severe mental illness in Beijing, China.

PubMed

Wang, Li; Zhang, Zhiguo; Yan, Qiuli; Lu, Jie; Gao, Baoyin; Zhao, Yanlin; Pang, Yu

2017-01-18

Although the prevalence of tuberculosis has decreased significantly over the past decades, the certain populations with mental illness are at increased risk for tuberculosis infection and transmission. However, no studies have examined the performance of different laboratory examination methods among people with severe mental illness in China. In this study, we firstly performed a retrospective study to evaluate the feasibility of three routine laboratory methods, including sputum microscopy, solid culture and GeneXpert, to diagnose tuberculosis patients with mental illness. During August 2010 and March 2013, a total of 251 TB patients based on clinical and radiographic criteria with severe mental illness were enrolled in this study. The majority of patients was homeless (97/251, 38.6%), and the other 62 (24.7%) and 92 (36.7%) were from urban and rural region, respectively. The most frequently diagnosed mental illness was schizophrenia, accounting for 84.1% (211/251) of patients available for analysis. In addition, the laboratory received 753 sputum samples collected from these 251 TB patients, of which 76.0% (572/753) of samples were classified as salivary sputum, which were unqualified for microscopy and culture. When the test results were analyzed by patients, the positive numbers of TB patients detected by sputum microscopy, solid culture and GeneXpert were 3 (1.2%), 5 (2.0%) and 5 (2.0%), respectively. In conclusion, our findings reveal that the current laboratory examinations based on sputum samples seem not to be suitable for the diagnosis of active TB in the persons with severe mental illness. The products using a non-invasive specimen such as urine deserve further evaluation, which may generate benefit for the early diagnosis of TB in this special population.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

PubMed Central

Stefano, Daniela De; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; Gregorio, Fabiola De; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; Rosa, Giuseppe De; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

2014-01-01

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in CftrF508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation. PMID:25350163

Prevention of exacerbations in patients with stable non-cystic fibrosis bronchiectasis: a systematic review and meta-analysis of pharmacological and non-pharmacological therapies.

PubMed

Abu Dabrh, Abd Moain; Hill, Adam T; Dobler, Claudia C; Asi, Noor; Farah, Wigdan H; Haydour, Qusay; Wang, Zhen; Benkhadra, Khalid; Prokop, Larry J; Murad, Mohammad Hassan

2018-04-20

Several pharmacological and non-pharmacological therapies are used to treat stable bronchiectasis of non-cystic fibrosis (CF) aetiology. We conducted a systematic review and meta-analysis to assess the evidence of the effectiveness of pharmacological and non-pharmacological treatment options in patients with stable non-CF bronchiectasis with a focus on reducing exacerbations. Multiple databases were searched through September 2017. Outcomes included the number of patients with exacerbation events, mean number of exacerbations, hospitalisations, mortality, quality of life measures, and safety and adverse effects. Meta-analysis was conducted using the random effects model. 30 randomised controlled trials enrolled subjects with non-CF bronchiectasis using different interventions. Moderate-quality evidence supported the effect of long-term antibiotics (≥3 months) on lowering the number of patients experiencing exacerbation events (relative risk 0.77 (95% CI 0.68 to 0.89)), reducing number of exacerbations (incidence rate ratio 0.62 (95% CI 0.49 to 0.78)), improving forced expiratory volume (litre) in the first second (FEV 1 ) (weighted mean difference (WMD); 0.02 (95% CI 0.00 to 0.04)), decreasing sputum purulence scores (numerical scale of 1-8) (WMD -0.90 (95% CI -1.58 to -0.22)) and improving quality of life scores assessed by the St George's Respiratory Questionnaire (WMD -6.07 (95% CI -10.7 to -1.43)). Bronchospasm increased with inhaled antibiotics while diarrhoea increased particularly with oral macrolide therapy. Moderate-quality evidence supports long-term antibiotic therapy for preventing exacerbations in stable non-CF bronchiectasis. However, data about the optimum agent, mode of therapy and length of treatment are limited. There is paucity of high-quality evidence to support the management of stable non-CF bronchiectasis including prevention of exacerbations. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

[Examination about utility of a Streptococcus pneumoniae capsular antigen swiftness search kit urine in a pneumonia patient].

PubMed

Hashikita, Giichi; Yamaguti, Toshiyuki; Tachi, Yoshimi; Kishi, Etsuko; Kawamura, Toru; Takahashi, Shun; Arai, Yukie; Koyama, Sachie; Huruhata, Toshihumi; Itabashi, Akira; Oka, Yoko; Yamazaki, Tsutomu; Maesaki, Sigefumi

2005-01-01

We investigated the usefullness of Binax NOW urine antigen test, an immunochromatographic assay that binds any soluble Streptococcus pneumoniae antigen (C polysaccharide) for the diagnosis of penumoniae form September 2003 to March 2005. We used 372 samples form the patinets with pneumoniae diagnosed for blood or sputum cultuter or gram-stained sputum smear. Out of 24 culture positive specimens, Binax NOW urine antigen test, showed positive in 18 (75%) specimens. The sensitivity of sputum and blood culture was 71.7% and 83.3%, respectively. Binax NOW urine antigen test was seemed false positives in 55 samples, false negatives in 6 samples. The specificity of Binax NOW urine antigen test was evaluated 84.1%. Overall agreement among tests was 83.6%. When compared to culture, false negative urine antigen may be the result of colonizing S. pneumoniae in sputum or pneumonia caused by an agent other than S. pneumoniae. CRP values for cases were both urine antigen and culture were positive ranged from 40 mg/dl to 10 mg/dl while urine antigen and culture negative cases were predominantly less than 10 mg/dl. Positive blood and pleural fluid culture cases were consistently associated with strongly positive urine antigen tests. Non-agreement between urine antigen, culture, and microscopy may be the result of specimen quality, labile nature of S. pneumoniae and antimicrobial therapy.

Application Values of T-SPOT.TB in Clinical Rapid Diagnosis of Tuberculosis.

PubMed

Zhu, Feng; Ou, Qinfang; Zheng, Jian

2018-01-01

This paper aims to explore the application value of tuberculosis-specific enzyme-linked immunospot assay (T-SPOT.TB) in the diagnosis of tuberculosis. Fifty one patients with tuberculosis (TB) admitted to Wuxi No.5 People's Hospital, Wuxi, China from June 2015 to June 2017 were selected as the TB group, and 40 patients without tuberculosis admitted in the same period were randomly selected as the non-TB group. Patients in the two groups received T-SPOT.TB, TB antibody (TB-Ab) test and mycobacterium TB deoxyribonucleic acid (TB-DNA) test, and the results were compared. Comparisons of the sensitivity of the three methods showed that the sensitivity of T-SPOT.TB was the highest, followed by TB-DNA from sputum samples, and that of TB-Ab was the lowest. The specificity of TB-Ab was the highest, followed by T-SPOT.TB, and that of TB-DNA from sputum samples was the lowest. In the receiver operating characteristic (ROC) curve analysis, the area under curve (AUC) of T-SPOT.TB (0.896) was the highest, followed by TB-DNA from sputum samples (0.772), and that of sputum smears (0.698) was the lowest. T-SPOT.TB can quickly and accurately determine the presence of tuberculosis infection, and it is a non-invasive examination, which can further assist in the diagnosis and guide the treatment.

Diagnostic accuracy, incremental yield and prognostic value of Determine TB-LAM for routine diagnostic testing for tuberculosis in HIV-infected patients requiring acute hospital admission in South Africa: a prospective cohort.

PubMed

Lawn, Stephen D; Kerkhoff, Andrew D; Burton, Rosie; Schutz, Charlotte; Boulle, Andrew; Vogt, Monica; Gupta-Wright, Ankur; Nicol, Mark P; Meintjes, Graeme

2017-03-21

We previously reported that one-third of HIV-positive adults requiring medical admission to a South African district hospital had laboratory-confirmed tuberculosis (TB) and that almost two-thirds of cases could be rapidly diagnosed using Xpert MTB/RIF-testing of concentrated urine samples obtained on the first day of admission. Implementation of urine-based, routine, point-of-care TB screening is an attractive intervention that might be facilitated by use of a simple, low-cost diagnostic tool, such as the Determine TB-LAM lateral-flow rapid test for HIV-associated TB. Sputum, urine and blood samples were systematically obtained from unselected HIV-positive adults within 24 hours of admission to a South African township hospital. Additional clinical samples were obtained during hospitalization as clinically indicated. TB was defined by the detection of Mycobacterium tuberculosis in any sample using Xpert MTB/RIF or liquid culture. The diagnostic yield, accuracy and prognostic value of urine-lipoarabinomannan (LAM) testing were determined, but urine-LAM results did not inform treatment decisions. Consecutive HIV-positive adult acute medical admissions not already receiving TB treatment (n = 427) were enrolled regardless of clinical presentation or symptoms. TB was diagnosed in 139 patients (TB prevalence 32.6%; median CD4 count 80 cells/μL). In the first 24 hours of admission, sputum (spot and/or induced) samples were obtained from 37.0% of patients and urine samples from 99.5% of patients (P < 0.001). The diagnostic yields from these specimens were 19.4% (n = 27/139) for sputum-microscopy, 26.6% (n = 37/139) for sputum-Xpert, 38.1% (n = 53/139) for urine-LAM and 52.5% (n = 73/139) for sputum-Xpert/urine-LAM combined (P < 0.01). Corresponding yields among patients with CD4 counts <100 cells/μL were 18.9%, 24.3%, 55.4% and 63.5%, respectively (P < 0.01). The diagnostic yield of urine-LAM was unrelated to respiratory symptoms, and LAM assay specificity (using a grade-2 cut-off) was 98.9% (274/277; 95% confidence interval [CI] 96.9-99.8). Among TB cases, positive urine-LAM status was strongly associated with mortality at 90 days (adjusted hazard ratio 4.20; 95% CI 1.50-11.75). Routine testing for TB in newly admitted HIV-positive adults using Determine TB-LAM to test urine provides major incremental diagnostic yield with very high specificity when used in combination with sputum testing and has important utility among those without respiratory TB symptoms and/or unable to produce sputum. The assay also rapidly identifies individuals with a poor prognosis.

2-Aminoacetophenone as a potential breath biomarker for Pseudomonas aeruginosa in the cystic fibrosis lung

PubMed Central

2010-01-01

Background Pseudomonas aeruginosa infections are associated with progressive life threatening decline of lung function in cystic fibrosis sufferers. Growth of Ps. aeruginosa releases a "grape-like" odour that has been identified as the microbial volatile organic compound 2-aminoacetophenone (2-AA). Methods We investigated 2-AA for its specificity to Ps. aeruginosa and its suitability as a potential breath biomarker of colonisation or infection by Solid Phase Micro Extraction and Gas Chromatography-Mass Spectrometry (GC/MS). Results Cultures of 20 clinical strains of Ps. aeruginosa but not other respiratory pathogens had high concentrations of 2-AA in the head space of in vitro cultures when analysed by GC/MS. 2-AA was stable for 6 hours in deactivated glass sampling bulbs but was not stable in Tedlar® bags. Optimisation of GC/MS allowed detection levels of 2-AA to low pico mol/mol range in breath. The 2-AA was detected in a significantly higher proportion of subjects colonised with Ps. aeruginosa 15/16 (93.7%) than both the healthy controls 5/17 (29%) (p < 0.0002) and CF patients not colonised with Ps. aeruginosa 4/13(30.7%) (p < 0.001). The sensitivity and specificity of the 2-AA breath test compared to isolation of Ps. aeruginosa in sputum and/or BALF was 93.8% (95% CI, 67-99) and 69.2% (95% CI, 38-89) respectively. The peak integration values for 2-AA analysis in the breath samples were significantly higher in Ps. aeruginosa colonised subjects (median 242, range 0-1243) than the healthy controls (median 0, range 0-161; p < 0.001) and CF subjects not colonised with Ps. aeruginosa (median 0, range 0-287; p < 0.003) Conclusions Our results report 2-AA as a promising breath biomarker for the detection of Ps. aeruginosa infections in the cystic fibrosis lung. PMID:21054900

Evaluation of OMNIgene®•SPUTUM for tuberculosis sample processing and liquid culture at a hospital laboratory in India.

PubMed

Nambiar, Remya; Shaw, Kaitlyn; Curry, Patricia S

2017-10-01

This preliminary evaluation examined the reagent OMNIgene®•SPUTUM (OM-S) as a tool to eliminate NaOH/NALC processing prior to Middlebrook liquid culture for Mycobacterium tuberculosis (MTb). Twenty-seven manually split samples (OM-S-treated vs. NaOH/NALC) showed 100% agreement: 81.5% MTb-positive and 18.5% MTb-negative. On average, OM-S-treated specimens required 1.2 additional days to culture positivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

PubMed

Conese, Massimo; Castellani, Stefano; Lepore, Silvia; Palumbo, Orazio; Manca, Antonio; Santostasi, Teresa; Polizzi, Angela Maria; Copetti, Massimiliano; Di Gioia, Sante; Casavola, Valeria; Guerra, Lorenzo; Diana, Anna; Montemurro, Pasqualina; Mariggiò, Maria Addolorata; Gallo, Crescenzio; Maffione, Angela Bruna; Carella, Massimo

2014-01-01

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.

Evaluation of Genome-Wide Expression Profiles of Blood and Sputum Neutrophils in Cystic Fibrosis Patients Before and After Antibiotic Therapy

PubMed Central

Conese, Massimo; Castellani, Stefano; Lepore, Silvia; Palumbo, Orazio; Manca, Antonio; Santostasi, Teresa; Polizzi, Angela Maria; Copetti, Massimiliano; Di Gioia, Sante; Casavola, Valeria; Guerra, Lorenzo; Diana, Anna; Montemurro, Pasqualina; Mariggiò, Maria Addolorata; Gallo, Crescenzio; Maffione, Angela Bruna; Carella, Massimo

2014-01-01

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to “healthy” condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis. PMID:25084273

Factors Associated with Persistent Sputum Positivity at the End of the Second Month of Tuberculosis Treatment in Lithuania.

PubMed

Diktanas, Saulius; Vasiliauskiene, Edita; Polubenko, Katazyna; Danila, Edvardas; Celedinaite, Indre; Boreikaite, Evelina; Misiunas, Kipras

2018-06-19

Non-conversion of sputum smear and culture prolongs the infectivity of the patient and has been associated with unfavorable outcomes. We aimed to evaluate factors associated with persistent sputum positivity at the end of two months of treatment of new case pulmonary tuberculosis (TB). Data of 87 human immunodeficiency virus-negative patients with culture-positive drug-susceptible pulmonary TB admitted to local university hospital between September 2015 and September 2016 were reviewed. Factors associated with sputum smear and/or culture positivity at the end of the second month of treatment were analyzed. Twenty-two patients (25.3%) remained smear and/or culture-positive. Male sex, lower body mass index (BMI), unemployment, alcohol abuse, higher number of lobes involved and cavities on chest X-rays, shorter time to detection (TTD) on liquid cultures, higher respiratory sample smear grading and colony count in solid cultures, higher C-reactive protein, erythrocyte sedimentation rate, leukocytosis, thrombocytosis, and anemia were all significantly associated with persistent sputum positivity. However, in the logistic regression analysis only male sex, lower BMI, alcohol abuse, higher radiological involvement, cavitation, higher smear grading, higher colony count in solid cultures and shorter TTD were determined as independent factors associated with persistent sputum positivity at the end of 2 months of treatment. In conclusion, higher sputum smear and culture grading at diagnosis, shorter TTD, higher number of lobes involved, cavitation, male sex, alcohol abuse, and lower BMI were independently associated with persistent sputum positivity. These factors should be sought when distinguishing which patients will remain infectious longer and possibly have worse outcomes. Copyright©2018. The Korean Academy of Tuberculosis and Respiratory Diseases.

Evaluation of Mycobacterium tuberculosis viability in OMNIgene-SPUTUM reagent upon multi-day transport at ambient temperature.

PubMed

Tagliani, Elisa; Alagna, Riccardo; Tafaj, Silva; Hafizi, Hasan; Cirillo, Daniela Maria

2017-10-02

Maintaining the quality of clinical specimens for tuberculosis (TB) testing is a major challenge in many high TB burden-limited resources countries. Sample referral systems in low and middle income countries are often weak and the maintenance of the cold-chain challenging and very costly for TB programs. The development of transport media allowing the preservation of samples without refrigeration is critical for increasing access to TB diagnostic services and for reducing the costs related to testing. We evaluated the performance of OMNIgene-SPUTUM (OM-S) reagent for the maintenance of Mycobacterium tuberculosis (MTB) viability in sputum samples in the absence of refrigeration and its capacity to stabilize nucleic acid for molecular testing. A total of 329 sputum specimens from presumptive TB cases collected at the National Reference Laboratory in Tirana, Albania, were either decontaminated by a conventional method or processed with OM-S reagent and stored at room temperature. Samples in OM-S were shipped to the Supranational Reference Laboratory in Milan, Italy, at various times and processed for liquid culture. Our data show that OM-S maintains MTB viability for at least three weeks in the absence of refrigeration and improves the quality of culture resulting in a contamination rate lower than 0.5%. However, a significant delay in the time to culture positivity was observed for samples stored for more than two weeks in OM-S. Overall, OM-S offers multiple benefits both at laboratory and TB national program level by increasing the availability to quality diagnostics, promoting access to health care services and strengthening TB patient care especially in hard to reach populations.

Long-term follow-up of distal intestinal obstruction syndrome in cystic fibrosis.

PubMed

Lavie, Moran; Manovitz, Tzipora; Vilozni, Daphna; Levy-Mendelovich, Sarina; Sarouk, Ifat; Weintraubv, Ilana; Shoseyov, David; Cohen-Cymberknoh, Malena; Rivlin, Joseph; Efrati, Ori

2015-01-07

To investigate the long-term follow-up of distal intestinal obstruction syndrome (DIOS) in Israeli cystic fibrosis (CF) patients. This is a multi-center, comparative, retrospective study in which we reviewed the medical records of all CF patients from three major CF centers in Israel who were treated in the period from 1980 to 2012. Patients diagnosed with DIOS were defined as the study group. The patients were diagnosed with DIOS based on their clinical presentation and typical findings on either abdominal X-ray or computerized tomography scan. For the control group, CF patients with no DIOS were matched to the patients in the study group for age, sex, and cystic fibrosis transmembrane conductance regulator (CFTR) mutations. For both groups, the collected data included age, sex, CFTR genotype, weight, height, and body mass index. Clinical data included respiratory function tests in the last five years prior to the study, respiratory function test immediately before and after the DIOS event, number of hospitalizations, sputum culture results, and CF-related conditions diagnosed according to the CF clinical practice guidelines. In the study group, data on the DIOS treatment and tendency for DIOS recurrence were also analyzed. The medical charts for a total of 350 CF patients were reviewed. Of the 350 CF patients, 26 (7.4%) were diagnosed with DIOS. The control group included 31 CF patients with no DIOS diagnosis. The mean follow-up period was 21.6 ± 8.2 years. The total of DIOS episodes in the follow-up period was 60. The distribution of DIOS episodes was as follows: 6/26 (23.1%) study patients had one episode of DIOS in their lifetime, 7/26 (26.9%) had two episodes, 7/26 (26.9%) had three episodes, and 6/26 (23.1%) had four or more episodes. Compared to the control group, DIOS patients had a significantly higher incidence of meconium ileus in the past (65.4% vs 0%, respectively, P < 0.02), more Aspergillus spp. colonization (34.6% vs 3.2%, respectively, P < 0.02), and a higher number of hospitalizations due to respiratory exacerbations (8.6 vs 6.2 mean total hospitalizations per follow-up period, respectively, P < 0.02). No other significant differences were found between the control and study groups. The conservative treatment of DIOS, which mainly includes hydration and stool softeners, was successful in 82% of the episodes. The survival rate was similar for both groups. CF patients with DIOS suffer from recurrent hospitalizations and airway pathogen acquisition. Although recurrence of DIOS is common, conservative treatment is successful in most patients.

Induced sputum deposition improves diagnostic yields of pulmonary alveolar proteinosis: A clinicopathological and methodological study of 17 cases.

PubMed

Huang, Ziling; Yi, Xianghua; Luo, Benfang; Zhu, Jian; Wu, Yunjin; Jiang, Wenxia; Chu, Haiqing; Yang, Zhongmin; Li, Shuai; Zhu, Hailong; Zhang, Suxia; Zhang, Lanjing; Zeng, Yu

2016-01-01

Pulmonary alveolar proteinosis (PAP) is a rare diffuse lung disease characterized by the accumulation of intra-alveolar lipoprotein-like surfactants. Lung core biopsy and bronchoalveolar lavage (BAL) fluid are currently the two major sources of sampling for diagnosis. In the present study, we assessed the value of induced sputum in diagnosing PAP by transmission electron microscopy and examined the PAP 2-year death rate in Asians. Transmission electron microscopy was performed on the samples from 17 patients with PAP, 13 patients with inflammatory lung diseases, and 13 healthy adults. The PAP patients were followed up for 3-156 months, and inflammatory lung diseases patients or healthy adults for 12-36 months. The ultrastructural features including diagnostic lamellar bodies of induced sputum deposition (ISD) samples were similar to that of the BAL fluid sediment. However, the rates of lamellar bodies were 73.7% in the ISD group, significantly higher than the spontaneous sputum deposition (SSD) group (42.1%, P < .0487) and similar to the BAL sediment (76.2%) and the lung biopsy (54.5%) groups. The overall 2-year death rate of our PAP patients was 17.6% (3/17), not statistically different from the healthy adults and patients with inflammatory diseases (0/13, P = .237 for both). ISD may be the preferred non-invasive sampling method for diagnosing PAP by electronic microscopy because of the higher diagnostic yield than SSD. The diagnostic yields of this noninvasive method were similar to that of lung core biopsy and BAL.

Synthesis and electrochemical detection of a thiazolyl-indole natural product isolated from the nosocomial pathogen Pseudomonas aeruginosa.

PubMed

Buzid, Alyah; Muimhneacháin, Eoin Ó; Reen, F Jerry; Hayes, Phyllis E; Pardo, Leticia M; Shang, Fengjun; O'Gara, Fergal; Sperry, Jonathan; Luong, John H T; Glennon, Jeremy D; McGlacken, Gerard P

2016-09-01

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, capable of surviving in a broad range of natural environments and quickly acquiring resistance. It is associated with hospital-acquired infections, particularly in patients with compromised immunity, and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is also of nosocomial importance on dairy farms and veterinary hospitals, where it is a key morbidity factor in bovine mastitis. P. aeruginosa uses a cell-cell communication system consisting of signalling molecules to coordinate bacterial secondary metabolites, biofilm formation, and virulence. Simple and sensitive methods for the detection of biomolecules as indicators of P. aeruginosa infection would be of great clinical importance. Here, we report the synthesis of the P. aeruginosa natural product, barakacin, which was recently isolated from the bovine ruminal strain ZIO. A simple and sensitive electrochemical method was used for barakacin detection using a boron-doped diamond (BDD) and glassy carbon (GC) electrodes, based on cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The influence of electrolyte pH on the peak potential and peak currents was also investigated. At pH 2.0, the peak current was linearly dependent on barakacin concentration (in the range used, 1-10 μM), with correlation coefficients greater than 0.98 on both electrodes. The detection limit (S/N = 3) on the BDD electrode was 100-fold lower than that obtained on the GC electrode. The optimized method using the BDD electrode was extended to bovine (cow feces) and human (sputum of a CF patient) samples. Spiked barakacin was easily detected in these matrices at a limit of 0.5 and 0.05 μM, respectively. Graphical abstract Electrochemical detection of barakacin.

Complex sputum microbial composition in patients with pulmonary tuberculosis

PubMed Central

2012-01-01

Background An increasing number of studies have implicated the microbiome in certain diseases, especially chronic diseases. In this study, the bacterial communities in the sputum of pulmonary tuberculosis patients were explored. Total DNA was extracted from sputum samples from 31 pulmonary tuberculosis patients and respiratory secretions of 24 healthy participants. The 16S rRNA V3 hyper-variable regions were amplified using bar-coded primers and pyro-sequenced using Roche 454 FLX. Results The results showed that the microbiota in the sputum of pulmonary tuberculosis patients were more diverse than those of healthy participants (p<0.05). The sequences were classified into 24 phyla, all of which were found in pulmonary tuberculosis patients and 17 of which were found in healthy participants. Furthermore, many foreign bacteria, such as Stenotrophomonas, Cupriavidus, Pseudomonas, Thermus, Sphingomonas, Methylobacterium, Diaphorobacter, Comamonas, and Mobilicoccus, were unique to pulmonary tuberculosis patients. Conclusions This study concluded that the microbial composition of the respiratory tract of pulmonary tuberculosis patients is more complicated than that of healthy participants, and many foreign bacteria were found in the sputum of pulmonary tuberculosis patients. The roles of these foreign bacteria in the onset or development of pulmonary tuberculosis shoud be considered by clinicians. PMID:23176186

Comparison of a new gold immunochromatographic assay for the rapid diagnosis of the novel influenza A (H7N9) virus with cell culture and a real-time reverse-transcription PCR assay.

PubMed

Jin, Changzhong; Wu, Nanping; Peng, Xiaorong; Yao, Hangping; Lu, Xiangyun; Chen, Yu; Wu, Haibo; Xie, Tiansheng; Cheng, Linfang; Liu, Fumin; Kang, Keren; Tang, Shixing; Li, Lanjuan

2014-01-01

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings.

Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay

PubMed Central

Wu, Nanping; Peng, Xiaorong; Yao, Hangping; Lu, Xiangyun; Chen, Yu; Wu, Haibo; Xie, Tiansheng; Cheng, Linfang; Liu, Fumin; Kang, Keren; Tang, Shixing; Li, Lanjuan

2014-01-01

We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings. PMID:24822207

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

2016-05-31

The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information.

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients

PubMed Central

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

2016-01-01

Background The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. Results ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). Methods A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). Conclusions We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information. PMID:27102299

Volumetric capnography for the evaluation of pulmonary disease in adult patients with cystic fibrosis and noncystic fibrosis bronchiectasis.

PubMed

Veronez, L; Moreira, M M; Soares, S T P; Pereira, M C; Ribeiro, M A G O; Ribeiro, J D; Terzi, R G G; Martins, L C; Paschoal, I A

2010-06-01

This study was designed to use volumetric capnography to evaluate the breathing pattern and ventilation inhomogeneities in patients with chronic sputum production and bronchiectasis and to correlate the phase 3 slope of the capnographic curve to spirometric measurements. Twenty-four patients with cystic fibrosis (CF) and 21 patients with noncystic fibrosis idiopathic bronchiectasis (BC) were serially enrolled. The diagnosis of cystic fibrosis was based on the finding of at least two abnormal sweat chloride concentrations (iontophoresis sweat test). The diagnosis of bronchiectasis was made when the patient had a complaint of chronic sputum production and compatible findings at high-resolution computed tomography (HRCT) scan of the thorax. Spirometric tests and volumetric capnography were performed. The 114 subjects of the control group for capnographic variables were nonsmoker volunteers, who had no respiratory symptoms whatsoever and no past or present history of lung disease. Compared with controls, patients in CF group had lower SpO(2) (P < 0.0001), higher respiratory rates (RR) (P < 0.0001), smaller expiratory volumes normalized for weight (V(E)/kg) (P < 0.028), smaller expiratory times (Te) (P < 0.0001), and greater phase 3 Slopes normalized for tidal volume (P3Slp/V(E)) (P < 0.0001). Compared with controls, patients in the BC group had lower SpO(2) (P < 0.0001), higher RR (P < 0.004), smaller V(E)/kg (P < 0.04), smaller Te (P < 0.007), greater P3Slp/V(E) (P < 0.0001), and smaller VCO(2) (P < 0.0002). The pooled data from the two patient groups compared with controls showed that the patients had lower SpO(2) (P < 0.0001), higher RR (P < 0.0001), smaller V(E)/kg (P < 0.05), smaller Te (P < 0.0001), greater P3Slp/V(E) (P < 0.0001), and smaller VCO(2) (P < 0.0003). All of the capnographic and spirometric variables evaluated showed no significant differences between CF and BC patients. Spirometric data in this study reveals that the patients had obstructive defects with concomitant low vital capacities and both groups had very similar abnormalities. The capnographic variables in the patient group suggest a restrictive respiratory pattern (greater respiratory rates, smaller expiratory times and expiratory volumes, normal peak expiratory flows). Both groups of patients showed increased phase III slopes compared with controls, which probably indicates the presence of diffuse disease of small airways in both conditions leading to inhomogeneities of ventilation.

Breath-based biomarkers for tuberculosis

NASA Astrophysics Data System (ADS)

Kolk, Arend H. J.; van Berkel, Joep J. B. N.; Claassens, Mareli M.; Walters, Elisabeth; Kuijper, Sjoukje; Dallinga, Jan W.; van Schooten, Fredrik-Jan

2012-06-01

We investigated the potential of breath analysis by gas chromatography - mass spectrometry (GC-MS) to discriminate between samples collected prospectively from patients with suspected tuberculosis (TB). Samples were obtained in a TB endemic setting in South Africa where 28% of the culture proven TB patients had a Ziehl-Neelsen (ZN) negative sputum smear. A training set of breath samples from 50 sputum culture proven TB patients and 50 culture negative non-TB patients was analyzed by GC-MS. A classification model with 7 compounds resulted in a training set with a sensitivity of 72%, specificity of 86% and accuracy of 79% compared with culture. The classification model was validated with an independent set of breath samples from 21 TB and 50 non-TB patients. A sensitivity of 62%, specificity of 84% and accuracy of 77% was found. We conclude that the 7 volatile organic compounds (VOCs) that discriminate breath samples from TB and non-TB patients in our study population are probably host-response related VOCs and are not derived from the VOCs secreted by M. tuberculosis. It is concluded that at present GC-MS breath analysis is able to differentiate between TB and non-TB breath samples even among patients with a negative ZN sputum smear but a positive culture for M. tuberculosis. Further research is required to improve the sensitivity and specificity before this method can be used in routine laboratories.

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples.

PubMed

Strålin, Kristoffer; Bäckman, Anders; Holmberg, Hans; Fredlund, Hans; Olcén, Per

2005-02-01

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.

Pneumocystis jiroveci in HIV/AIDS patients: detection by FTA filter paper together with PCR in noninvasive induced sputum specimens.

PubMed

Jaijakul, Siraya; Saksirisampant, Wilai; Prownebon, Juraratt; Yenthakam, Sutin; Mungthin, Mathirut; Leelayoova, Saovanee; Nuchprayoon, Surang

2005-09-01

To detect P. jiroveci (previously named P. carinii) by PCR using FTA filter paper to extract the DNA, from noninvasive induced sputum samples of HIV/AIDS patients. Fifty two HIV/AIDS patients suspected of Pneumocystis jiroveci pneumonia (PJP) in King Chulalongkorn Memorial Hospital were recruited. Both cytological method and PCR with FTA filter paper technique were performed to detect P jiroveci from each specimen. The detectability rate of P. jiroveci infection was 21%. The PCR with FTA filter paper method was 4 folds much more sensitive than Giemsa staining technique. P. jiroveci was detected in 18% of the HIV/AIDS patients in spite of receiving standard PJP prophylaxis. Detection of P. jiroveci by using FTA filter paper together with PCR in induced sputum samples could detect more cases of P. jiroveci infection than by using cytological method. DNA extraction using the FTA filter paper was more rapid and convenient than other extraction methods. The causes of failure of PJP prophylaxis should be evaluated.

Efficient zinc uptake is critical for the ability of Pseudomonas aeruginosa to express virulence traits and colonize the human lung.

PubMed

Mastropasqua, Maria Chiara; Lamont, Iain; Martin, Lois W; Reid, David W; D'Orazio, Melania; Battistoni, Andrea

2018-07-01

We have recently shown that Pseudomonas aeruginosa, an opportunistic pathogen that chronically infects the lungs of patients with cystic fibrosis (CF) and other forms of lung disease, is extremely efficient in recruiting zinc from the environment and that this capability is required for its ability to cause acute lung infections in mice. To verify that P. aeruginosa faces zinc shortage when colonizing the lungs of human patients, we analyzed the expression of three genes that are highly induced under conditions of zinc deficiency (zrmA, dksA2 and rpmE2), in bacteria in the sputum of patients with inflammatory lung disease. All three genes were expressed in all the analyzed sputum samples to a level much higher than that of bacteria grown in zinc-containing laboratory medium, supporting the hypothesis that P. aeruginosa is under zinc starvation during lung infections. We also found that the expression of several virulence traits that play a central role in the ability of P. aeruginosa to colonize the lung is affected by disruption of the most important zinc importing systems. Virulence features dependent on zinc intake include swarming and swimming motility and the ability to form biofilms. Furthermore, alterations in zinc assimilation interfere with the synthesis of the siderophore pyoverdine, suggesting that zinc recruitment could modulate iron uptake and affect siderophore-mediated cell signaling. Our results reveal that zinc uptake is likely to play a key role in the ability of P. aeruginosa to cause chronic lung infections and strongly modulates critical virulence traits of the pathogen. Taking into account the recent discovery that zinc uptake in P. aeruginosa is promoted by the release of a small molecular weight molecule showing high affinity for zinc, our data suggest novel and effective possibilities to control lung infections by these bacteria. Copyright © 2018 Elsevier GmbH. All rights reserved.

Novel approaches in diagnosing tuberculosis

NASA Astrophysics Data System (ADS)

Kolk, Arend H. J.; Dang, Ngoc A.; Kuijper, Sjoukje; Gibson, Tim; Anthony, Richard; Claassens, Mareli M.; Kaal, Erwin; Janssen, Hans-Gerd

2011-06-01

The WHO declared tuberculosis (TB) a global emergency. An estimated 8-9 million new cases occur each year with 2-3 million deaths. Currently, TB is diagnosed mostly by chest-X ray and staining of the mycobacteria in sputum with a detection limit of 1x104 bacteria /ml. There is an urgent need for better diagnostic tools for TB especially for developing countries. We have validated the electronic nose from TD Technology for the detection of Mycobacterium tuberculosis by headspace analysis of 284 sputum samples from TB patients. We used linear discriminant function analysis resulting in a sensitivity of 75% a specificity of 67% and an accuracy of 69%. Further research is still required to improve the results by choosing more selective sensors and sampling techniques. We used a fast gas chromatography- mass spectrometry method (GC-MS). The automated procedure is based on the injection of sputum samples which are methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM-GC-MS). Hexacosanoic acid in combination with tuberculostearic acid was found to be specific for the presence of M. tuberculosis. The detection limit was similar to microscopy. We found no false positives, all microscopy and culture positive samples were also found positive with the THM-GC-MS method. The detection of ribosomal RNA from the infecting organism offers great potential since rRNA molecules outnumber chromosomal DNA by a factor 1000. It thus may possible to detect the organism without amplification of the nucleic acids (NA). We used a capture and a tagged detector probe for the direct detection of M. tuberculosis in sputum. So far the detection limit is 1x106 bacteria / ml. Currently we are testing a Lab-On-A-Chip Interferometer detection system.

Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum.

PubMed

Poudel, A; Pandey, B D; Lekhak, B; Rijal, B; Sapkota, B R; Suzuki, Y

2009-01-01

Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis. Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (chi(2)=5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal.

Bronchial inflammation in seasonal allergic rhinitis with or without asthma in relation to natural exposure to pollen allergens.

PubMed

Panzner, P; Malkusová, I; Vachová, M; Liška, M; Brodská, P; Růžičková, O; Malý, M

2015-01-01

Nasal inflammation in allergic rhinitis enhances bronchial Th2 driven inflammation and development of asthma. We assessed bronchial inflammation induced by natural allergen exposure during pollen season in patients with pollinosis with or without asthma to show the intensity of inflammation in asthma and rhinitis and possible persistence of inflammation in periods without allergen exposure. Sputum was induced in 52 patients with seasonal allergic rhinitis without asthma, 38 patients with seasonal allergic rhinitis and seasonal asthma and 23 healthy volunteers. Sampling was performed 6-8 weeks before the expected beginning of symptoms, during symptomatic period and 6-8 weeks after the end of symptoms. Sputum ECP was measured by means of chemi-luminiscent immunometric assay and sputum cell counts were assessed by classical staining and immunocytochemistry. Sputum eosinophils were on the whole higher in both asthma and rhinitis compared to controls (p<0.001, p=0.003). The rise of eosinophils during pollen season compared with values out of pollen season was significant in asthma (classical staining) (p=0.014) and slightly apparent in rhinitis (immunocytochemistry) (p=0.073). The seasonal rise of sputum ECP was observed only in rhinitis (p=0.006). Inflammation of the lower airway in patients with allergic rhinitis with and without asthma has been confirmed by means of both sputum eosinophil count and sputum ECP level. Persistent inflammation of lower airway in periods without allergen exposure was proven in seasonal asthma. This may have implications for the therapy of seasonal allergic rhinitis with and without asthma in terms of promoting long-term anti-inflammatory treatment. Copyright © 2013 SEICAP. Published by Elsevier Espana. All rights reserved.

Sputum fungal smear

MedlinePlus

... in the test sample. Some labs use different measurements or test different samples. Talk to your doctor ... A.D.A.M. follows rigorous standards of quality and accountability. A.D.A.M. is among ...

Effects of overnight refrigeration on the microscopic evaluation of sputum.

PubMed Central

Penn, R L; Silberman, R

1984-01-01

Microscopic evaluation of sputum permits selection of specimens suitable for culture, assessment of likely pathogens, and the best interpretation of culture results. We prospectively evaluated 50 sputum specimens which were promptly submitted to our clinical laboratory; smears and cultures were performed both immediately and after 20 h of refrigeration. Specimens were grouped according to the numbers of squamous epithelial cells and neutrophils per low-power field present on coded Gram-stained smears. The numbers of bacteria in five oil immersion fields were used to characterize smears for predominant, mixed, or scanty forms. After refrigeration, only three specimens changed group from a definite loss of squamous epithelial cells, and only two changed group from a definite loss of neutrophils. Based on cellular composition, the majority of samples would have been processed identically both before and after refrigeration. In contrast, organism forms detected on smears and their relative quantities were dramatically altered after refrigeration. A predominant smear form was gained in 11 and lost in 8 refrigerated specimens. The frequent changes on smears observed overall resulted from both increases and decreases in numbers of bacteria and yeasts. The majority of sputum culture results were insignificantly affected by the refrigeration of specimens. We conclude that 20 h of refrigeration renders sputum useless for the microscopic evaluation of potential pathogens and the subsequent interpretation of culture results. However, overnight refrigeration does not affect the determination from smears of sputum suitability for culture based on cellular composition. PMID:6699145

Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

PubMed

Barletta, Francesca; Vandelannoote, Koen; Collantes, Jimena; Evans, Carlton A; Arévalo, Jorge; Rigouts, Leen

2014-10-01

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture. © The American Society of Tropical Medicine and Hygiene.

Performance of the TB-LAMP diagnostic assay in reference laboratories: Results from a multicentre study.

PubMed

Pham, Thu Hang; Peter, Jonathan; Mello, Fernanda C Q; Parraga, Tommy; Lan, Nguyen Thi Ngoc; Nabeta, Pamela; Valli, Eloise; Caceres, Tatiana; Dheda, Keertan; Dorman, Susan E; Hillemann, Doris; Gray, Christen M; Perkins, Mark D

2018-03-01

To evaluate the diagnostic performance of TB-LAMP, a manual molecular tuberculosis (TB) detection method, and provide comparison to the Xpert MTB/RIF assay. In a large multicentre study, two sputum samples were collected from participants with TB symptoms in reference laboratories in Peru, South Africa, Brazil, and Vietnam. Each sample was tested with TB-LAMP. The reference standard consisted of four direct smears, four cultures, and clinical and radiological findings. Individuals negative on conventional tests were followed up after 8 weeks. The Xpert MTB/RIF assay was performed on fresh or frozen samples as a molecular test comparison. A total of 1036 adults with suspected TB were enrolled. Among 375 culture-confirmed TB cases with 750 sputum samples, TB-LAMP detected 75.6% (95% confidence interval (CI) 71.8-79.4%), including 97.9% (95% CI 96.4-99.4%) of smear-positive TB samples and 46.6% (95% CI 40.6-52.7%) of smear-negative TB samples. Specificity in 477 culture-negative participants not treated for TB (954 sputum samples) was 98.7% (95% CI 97.9-99.6%). TB-LAMP test results were indeterminate in 0.3% of cases. TB-LAMP detects nearly all smear-positive and half of smear-negative TB cases and has a high specificity when performed in reference laboratories. Performance was similar to the Xpert MTB/RIF assay. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Anisotropic tubular filtering for automatic detection of acid-fast bacilli in Ziehl-Neelsen stained sputum smear samples

NASA Astrophysics Data System (ADS)

Raza, Shan-e.-Ahmed; Marjan, M. Q.; Arif, Muhammad; Butt, Farhana; Sultan, Faisal; Rajpoot, Nasir M.

2015-03-01

One of the main factors for high workload in pulmonary pathology in developing countries is the relatively large proportion of tuberculosis (TB) cases which can be detected with high throughput using automated approaches. TB is caused by Mycobacterium tuberculosis, which appears as thin, rod-shaped acid-fast bacillus (AFB) in Ziehl-Neelsen (ZN) stained sputum smear samples. In this paper, we present an algorithm for automatic detection of AFB in digitized images of ZN stained sputum smear samples under a light microscope. A key component of the proposed algorithm is the enhancement of raw input image using a novel anisotropic tubular filter (ATF) which suppresses the background noise while simultaneously enhancing strong anisotropic features of AFBs present in the image. The resulting image is then segmented using color features and candidate AFBs are identified. Finally, a support vector machine classifier using morphological features from candidate AFBs decides whether a given image is AFB positive or not. We demonstrate the effectiveness of the proposed ATF method with two different feature sets by showing that the proposed image analysis pipeline results in higher accuracy and F1-score than the same pipeline with standard median filtering for image enhancement.

Nuclear morphology for the detection of alterations in bronchial cells from lung cancer: an attempt to improve sensitivity and specificity.

PubMed

Fafin-Lefevre, Mélanie; Morlais, Fabrice; Guittet, Lydia; Clin, Bénédicte; Launoy, Guy; Galateau-Sallé, Françoise; Plancoulaine, Benoît; Herlin, Paulette; Letourneux, Marc

2011-08-01

To identify which morphologic or densitometric parameters are modified in cell nuclei from bronchopulmonary cancer based on 18 parameters involving shape, intensity, chromatin, texture, and DNA content and develop a bronchopulmonary cancer screening method relying on analysis of sputum sample cell nuclei. A total of 25 sputum samples from controls and 22 bronchial aspiration samples from patients presenting with bronchopulmonary cancer who were professionally exposed to cancer were used. After Feulgen staining, 18 morphologic and DNA content parameters were measured on cell nuclei, via image cytom- etry. A method was developed for analyzing distribution quantiles, compared with simply interpreting mean values, to characterize morphologic modifications in cell nuclei. Distribution analysis of parameters enabled us to distinguish 13 of 18 parameters that demonstrated significant differences between controls and cancer cases. These parameters, used alone, enabled us to distinguish two population types, with both sensitivity and specificity > 70%. Three parameters offered 100% sensitivity and specificity. When mean values offered high sensitivity and specificity, comparable or higher sensitivity and specificity values were observed for at least one of the corresponding quantiles. Analysis of modification in morphologic parameters via distribution analysis proved promising for screening bronchopulmonary cancer from sputum.

Vitamin C supplementation improve the sputum conversion culture rate in pulmonary tuberculosis treatment while rifampicin susceptible

NASA Astrophysics Data System (ADS)

Susanto, L.; Siregar, Y.; Kusumawati, L.

2018-03-01

The failure of first-line tuberculosis treatment greatly affects multiple drug-resistant tuberculosis. In vitro study of vitamin C induces the death of M. tuberculosis bacteria and accelerates healing of tuberculosis, so the multiple drug-resistant tuberculosis can be avoided. This research aimed to identify the effect of vitamin C as a supportive treatment on the sputum conversion rate. The randomizedand double group with a parallel design by matching pair method was used to collect samples. The first group was treated with standard tuberculosis treatment, and the other was given vitamin C supplementation. Vitamin C plasma level analyzation was performed before and after two months of treatment. Sputum conversion was evaluated every week for eight weeks. The comparison of vitamin C plasma level in pre and post-treatment group was significant (p=0.03) but not in the other group. There was no significant difference in vitamin C plasma level between two groups (p=0.21). The proportion of sputum conversion rate in both group in the first week was 0% vs. 9.6% (p=0.83) and the last week of study was 83.9% vs. 100% (p=0.02). In conclusion, vitamin C supplementation has effects in improving the healing process of tuberculosis patients as indicated by higher in sputum conversion rate.

Towards a versatile technique for tracking nanoparticle-mucus interaction: a step on the road

NASA Astrophysics Data System (ADS)

Nafee, N.; Schneider, M.

2014-02-01

Respiratory mucus is one of the main barriers for nanoparticle-based pulmonary delivery systems. This holds true especially for lung diseases like cystic fibrosis, where a very tenacious thick mucus layer hinders particle diffusion to the lung epithelium or the target area. Typically, mean square displacement of particles is used for mobility evaluation. In contrast, our objective is to develop a feasible technique to track directed particle penetration as a prerequisite for efficient pulmonary nanotherapy. Therefore, particle diffusion in artificial mucus was monitored based on confocal laser scanning microscopy (CLSM) and particle-mucus interaction was observed. As pharmaceutical relevant and benign materials, solid lipid nanoparticles (SLNs) were prepared by hot-melt emulsification using glyceryl behenate and different stabilizing agents such as poloxamer-407, tween-80, and polyvinyl alcohol (PVA). The diffusion of labeled SLNs in stained artificial sputum representing CF-patient sputum was verified by 3D time laps imaging. Thus, the effect of coating, particle size and mucus viscosity on nanoparticle diffusion was studied. Using image analysis software "Image J", the total fluorescent signal after 30 min in case of poloxamer-coated SLNs was 5 and 100 folds higher than tween- and PVA-coated SLNs, respectively. Nevertheless, increasing mucus viscosity reduced the diffusion of tweencoated SLNs by a factor of 10. Studying particle-mucus interaction by CLSM can be considered a promising and versatile technique.

Cystic fibrosis screening using the College panel: platform comparison and lessons learned from the first 20,000 samples.

PubMed

Strom, Charles M; Huang, Donghui; Buller, Arlene; Redman, Joy; Crossley, Beryl; Anderson, Ben; Entwistle, Tom; Sun, Weimin

2002-01-01

To determine the accuracy of two commercially available kits for cystic fibrosis (CF) genotyping and determine allele frequencies for the ACMG/ACOG recommended mutations. A total of 1,040 consecutive analyses using Roche CF Gold Strips and the ABI CF Genotyper were performed. Subsequently we performed analyses of 20,103 samples. Both kits accurately determined CF genotypes. The I148T mutation was found >100 times more frequently in carrier screening than in CF patients. Asymptomatic patients were identified who are compound heterozygotes for delta F508 and I148T. Four of 13 patients heterozygous for delta F508 and the IVS8-5T polymorphism had some symptoms of CF. Accurate and timely analysis can be performed for the ACMG CF panel. I148T is a low penetrance CF allele.

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis *

PubMed Central

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

OBJECTIVE: To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). METHODS: This was a cross-sectional study involving adolescents and adults aged ≥ 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. RESULTS: We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. CONCLUSIONS: The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis. PMID:23670503

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.

PubMed

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). This was a cross-sectional study involving adolescents and adults aged > 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis.

Development and psychometric validation of a cystic fibrosis knowledge scale.

PubMed

Balfour, Louise; Armstrong, Michael; Holly, Crystal; Gaudet, Ena; Aaron, Shawn; Tasca, George; Cameron, William; Pakhale, Smita

2014-11-01

Well-developed and validated measures of cystic fibrosis (CF) knowledge are scarce. The purpose of the present study is to develop and validate a CF knowledge scale that is brief, easy to use, self-administered and demonstrates clinical utility. A comprehensive literature search generated a pool of scale items; an expert panel of CF team members reviewed and provided recommendations for item inclusion. A focus group of CF patients and family members (n = 12) then reviewed the items for face validity and reading clarity. To evaluate the validity and reliability of the newly developed CF knowledge scale, it was administered to several different samples including CF patients (n = 45), respirology patients (n = 100), health-care providers (n = 74) and university student samples (psychology students, n = 71; medical students, n = 36). Internal consistency of the scale was high, with an alpha coefficient for the overall sample of .95 (n = 326). The scale also demonstrated excellent construct validity. This study is an important first step in a line of research that aims to develop and empirically validate a psycho-educational adherence intervention for improving quality of life and treatment outcomes among adult CF patients. The CF knowledge scale has potential applications as a clinical teaching tool with patients and health-care providers and could be used as an outcome measure in CF educational intervention studies aimed at optimizing CF treatment knowledge, adherence and quality of life among CF patients. © 2014 Asian Pacific Society of Respirology.

Diagnostic performance of automated liquid culture and molecular line probe assay in smear-negative pulmonary tuberculosis.

PubMed

Kotwal, Aarti; Biswas, Debasis; Raghuvanshi, Shailendra; Sindhwani, Girish; Kakati, Barnali; Sharma, Shweta

2017-04-01

The diagnosis of smear-negative pulmonary tuberculosis (PTB) is particularly challenging, and automated liquid culture and molecular line probe assays (LPA) may prove particularly useful. The objective of our study was to evaluate the diagnostic potential of automated liquid culture (ALC) technology and commercial LPA in sputum smear-negative PTB suspects. Spot sputum samples were collected from 145 chest-symptomatic smear-negative patients and subjected to ALC, direct drug susceptibility test (DST) testing and LPA, as per manufacturers' instructions. A diagnostic yield of 26.2% was observed among sputum smear-negative TB suspects with 47.4% of the culture isolates being either INH- and/or rifampicin-resistant. Complete agreement was observed between the results of ALC assay and LPA except for two isolates which demonstrated sensitivity to INH and rifampicin at direct DST but were rifampicin-resistant in LPA. Two novel mutations were also detected among the multidrug isolates by LPA. In view of the diagnostic challenges associated with the diagnosis of TB in sputum smear-negative patients, our study demonstrates the applicability of ALC and LPA in establishing diagnostic evidence of TB.

Long-Term Storage at -80°C: Effect on Rate of Recovery of Mycobacterium tuberculosis From Direct Acid-Fast Bacilli Smear-Positive Sputum Samples.

PubMed

Shinu, Pottathil; AshokKumar Singh, Varsha; Nair, Anroop; Farooq, Rumana; Ishaq, Sheikh

2016-09-01

The aim of this study was to evaluate the difference in the rate of recovery of Mycobacterium tuberculosis (MTB) from routinely cultured sputum and long-term stored sputum specimens (at -80°C) using Löwenstein-Jensen (LJ) media, Mycobacterium Growth Indicator Tube (BBL MGIT(TM) ), and Middlebrook 7H11 (MB 7H11) agar. Direct acid-fast bacilli smear-positive sputum specimens (both before and after storage [n = 136]) were studied (after culturing on LJ media, BBL MGIT(TM) , and MB 7H11 agar) and the performances were compared. For the detection of MTB, BBL MGIT(TM) and MB 7H11 agar (before storage) demonstrated a sensitivity, specificity, positive predictive value (PPV) of 98.28%, 30.77%, 92.68%, 66.67%, and negative predictive value (NPV) of 97.41%, 30.77%, 92.62%, 57.14%, respectively, when compared to LJ media (before storage). Similarly, BBL MGIT(TM) and MB 7H11 agar (after storage) demonstrated a sensitivity, specificity, PPV, and NPV of 95.5%, 38.89%, 90.6%, 58.33%, and 95.5%, 66.67 %, 94.64%, 70.59%, respectively, when compared to LJ media (after storage) for the detection of MTB. None of the culture techniques independently (both before and after storage) detected growth of MTB from all the sputum specimens studied. However, BBL MGIT(TM) system and LJ media combination (both before and after storage) effectively detected the growth of MTB from sputum specimens when compared to other culture technique combinations. © 2015 Wiley Periodicals, Inc.

Rapid, Efficient Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis in Sputum by Microscopic Observation of Broth Cultures

PubMed Central

Caviedes, Luz; Lee, Tien-Shun; Gilman, Robert H.; Sheen, Patricia; Spellman, Emily; Lee, Ellen H.; Berg, Douglas E.; Montenegro-James, Sonia

2000-01-01

Inexpensive, rapid, and reliable methods of detecting infection by and drug susceptibility of Mycobacterium tuberculosis (MTB) are crucial to the control of tuberculosis. The novel microscopic observation broth-drug susceptibility assay (MODS) detects early growth of MTB in liquid medium, allowing more timely diagnosis and drug susceptibility testing. Sputum samples from hospitalized patients in Peru were analyzed by using stains, culture, and PCR. Sensitivity of MODS (92%) compared favorably with the most sensitive of the other culture methods (93%). Sputum samples positive for tuberculosis were tested for susceptibility to isoniazid and rifampin with the microwell alamar blue assay (MABA) and MODS. In 89% of cases, there was concordance between MODS and MABA. Of the diagnostic and susceptibility testing methods used, MODS yielded results most rapidly (median, 9.0 and 9.5 days, respectively). MODS is a rapid, inexpensive, sensitive, and specific method for MTB detection and susceptibility testing; it is particularly appropriate for use in developing countries burdened by significant infection rates and increasing numbers of multiple-drug-resistant cases. PMID:10699023

Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. The Tuberculosis Working Group in Peru.

PubMed

Caviedes, L; Lee, T S; Gilman, R H; Sheen, P; Spellman, E; Lee, E H; Berg, D E; Montenegro-James, S

2000-03-01

Inexpensive, rapid, and reliable methods of detecting infection by and drug susceptibility of Mycobacterium tuberculosis (MTB) are crucial to the control of tuberculosis. The novel microscopic observation broth-drug susceptibility assay (MODS) detects early growth of MTB in liquid medium, allowing more timely diagnosis and drug susceptibility testing. Sputum samples from hospitalized patients in Peru were analyzed by using stains, culture, and PCR. Sensitivity of MODS (92%) compared favorably with the most sensitive of the other culture methods (93%). Sputum samples positive for tuberculosis were tested for susceptibility to isoniazid and rifampin with the microwell alamar blue assay (MABA) and MODS. In 89% of cases, there was concordance between MODS and MABA. Of the diagnostic and susceptibility testing methods used, MODS yielded results most rapidly (median, 9.0 and 9.5 days, respectively). MODS is a rapid, inexpensive, sensitive, and specific method for MTB detection and susceptibility testing; it is particularly appropriate for use in developing countries burdened by significant infection rates and increasing numbers of multiple-drug-resistant cases.

[Preliminary establishment of cytological examination and the normal reference values for hypertonic saline solution-induced sputum of healthy children in Guangzhou].

PubMed

Chen, De-hui; Zhong, Guo-yu; Luo, Wei; Chen, Qiao-li; Chen, Ru-chong; Lin, Yu-neng; Pan, Xiao-an; Li, Jin-ying; Wu, Shang-zhi; Lai, Ke-fang; Zhong, Nan-shan

2012-07-01

To establish the method of cytological examination and the normal reference values for hypertonic saline solution-induced sputum of healthy children (age range from 5 to 15 years) with physical examination in Guangzhou. A total of 352 children, 5 to 15 years old, were enrolled from primary school and middle school in Guangzhou from January to December, 2010. All subjects completed a standardized questionnaire on the presence of respiratory, allergic symptoms and family history, the medical history and the physical examination was performed by doctors, lung function (forced expiratory volume at 1 s in predicted normal, FEV(1)%) was determined. There were 266 healthy children (137 males, 129 females) who were selected and undergone hypertonic saline solution induction of sputum, and cytological examination was performed. Hypertonic saline (5%) was nebulized and inhaled for 15 - 30 min. No expectoration within 30 min was defined as failure, and the procedure was terminated. The part of opaque and higher density sputum samples was detected by cytology. The proportion of neutrophils, lymphocytes, eosinophils, macrophages and monocytes was calculated. This study was approved by the institutional Ethics Review Committee of First Affiliated Hospital of Guangzhou Medical College. Informed consent was obtained from the legal guardians of all participants following a detailed description of the purpose and potential benefits of the study. There were 175 subjects' induced sputum specimens (175/266, 65.8%), non-qualified sputum samples were obtained from 16 of the subjects. The proportions of median (IQR) of lymphocytes were 0.012 (0.020), 95%CI were ranged from 0.015 to 0.022; neutrophils 0.207 (0.330), 95%CI 0.266 - 0.356 macrophages 0.761 (0.327), 95%CI 0.607 - 0.699; eosinophils 0.004 (0.019), 95%CI 0.013 - 0.022. There were no significant differences in proportions of cytological findings of female or male, different age groups and second-hand smoking or not (all P > 0.05). The incidence of adverse event was 4.40% (7/159). The method and the preliminary data may be used for research, diagnosis and treatment of patients with chronic cough and airway inflammation.

Persistence of ZIKV-RNA in the cellular fraction of semen is accompanied by a surrogate-marker of viral replication. Diagnostic implications for sexual transmission.

PubMed

Biava, Mirella; Caglioti, Claudia; Castilletti, Concetta; Bordi, Licia; Carletti, Fabrizio; Colavita, Francesca; Quartu, Serena; Nicastri, Emanuele; Iannetta, Marco; Vairo, Francesco; Liuzzi, Giuseppina; Taglietti, Fabrizio; Ippolito, Giuseppe; Capobianchi, Maria Rosaria; Lalle, Eleonora

2018-01-01

As asymptomatic infections represent 80% of ZIKV-infected individuals, sexual transmission is a rising concern. Recent studies highlighted a preferential association of ZIKV with the cellular fraction (CF) of different specimen types. Our aim was to evaluate the presence of ZIKV-RNA in different body fluids, focusing on semen specimens to assess the ZIKV-RNA content in either the unfractionated sample, its CF or seminal plasma (SP). In addition, to establish if the presence of ZIKV genome was associated with active virus replication, we measured the levels of negative-strand ZIKV-RNA. ZIKV total-RNA was detected in blood, urine and unfractionated semen, and neg-RNA in semen CF and SP samples longitudinally collected from two ZIKV-positive men followed at the National Institute for Infectious Diseases "L. Spallanzani", Italy. In both patients, ZIKV total-RNA was detected in CF with ct values always lower than in the corresponding unfractionated samples, and was observed even in the CF from negative unfractionated semen samples. In Patient 2, neg-RNA was also detected in CF, suggesting ongoing viral replication. Our results demonstrate higher clinical sensitivity of CF as compared to whole semen testing, emphasizing the need to extend ZIKV-RNA testing to CF, to rule out virus presence and the possible risk of sexual transmission.

Clinical characteristics and airway inflammation profile of COPD persistent sputum producers.

PubMed

Khurana, S; Ravi, A; Sutula, J; Milone, R; Williamson, R; Plumb, J; Vestbo, J; Singh, D

2014-12-01

COPD patients with chronic bronchitis include a subgroup with persistent sputum production on most or every day. We hypothesized that COPD patients with persistent sputum production have a different profile of airway inflammation, and more severe clinical characteristics. To compare the airway inflammation profile and clinical characteristics of COPD persistent and non-persistent sputum producers. COPD persistent sputum producers (n = 26) and non-persistent sputum producers (n = 26) underwent sputum induction and pulmonary function tests. Exacerbation history was recorded; the St. George's Respiratory Questionnaire, Modified Medical Research Council Dyspnoea scale and COPD Assessment Tool were completed. 33 COPD patients provided sputum for bacteriology. Persistent sputum producers had lower post-bronchodilator FEV1% predicted (p = 0.01), diffusion capacity (p = 0.04), 6 min walk test distance (p = 0.05), and higher closing volume (p = 0.01), BODE index (p = 0.01), rate of bacterial colonization (p = 0.004) and exacerbations (p = 0.03) compared to non-persistent sputum producers. The mean SGRQ and CAT scores were higher in persistent sputum producers (p = 0.01 and 0.03 respectively). Sputum neutrophil and eosinophil total cell counts were higher in persistent sputum producers (p = 0.02 and 0.05 respectively). Sputum levels of eotaxin (p = 0.02), MCP-1 (p = 0.02), TNF-α (p = 0.03) and IL-6 (p = 0.05) were higher in persistent sputum producers. COPD persistent sputum producers have more severe clinical characteristics and increased concentrations of some inflammatory mediators in the airways.

Reassessment of the positive predictive value and specificity of Xpert MTB/RIF: a diagnostic accuracy study in the context of community-wide screening for tuberculosis.

PubMed

Ho, Jennifer; Nguyen, Phuong Thi Bich; Nguyen, Thu Anh; Tran, Khoa Hien; Van Nguyen, Son; Nguyen, Nhung Viet; Nguyen, Hoa Binh; Luu, Khanh Boi; Fox, Greg J; Marks, Guy B

2016-09-01

Community-wide screening for tuberculosis with Xpert MTB/RIF as a primary screening tool overcomes some of the limitations of conventional screening. However, concerns exist about the low positive predictive value of this test in screening settings. We did a cross-sectional assessment of this diagnostic test to directly estimate the actual positive predictive value of Xpert MTB/RIF when used in the setting of community-wide screening for tuberculosis, and to draw an inference about the specificity of the test for tuberculosis detection. Field staff visited households in 60 randomly selected villages in Ca Mau province, Vietnam. We included people aged 15 years or older who provided written informed consent and were able to produce 0·5 mL or more of sputum, irrespective of reported symptoms. Participants were tested with Xpert MTB/RIF, then those with positive results had two further sputum samples tested for smear microscopy and culture, and underwent chest radiography at the provincial TB Health Center. The positive predictive value of Xpert MTB/RIF was compared against two reference standards for tuberculosis diagnosis-a positive sputum culture for Mycobacterium tuberculosis, and a positive sputum culture or a chest radiograph consistent with active pulmonary tuberculosis. We then calculated the specificity of Xpert MTB/RIF for tuberculosis detection on the basis of these positive predictive values and disease prevalence in this setting. 43 435 adults consented to screening with Xpert MTB/RIF. Sputum samples of 0·5 mL or greater were collected from 23 202 participants, producing 22 673 valid results. 169 participants had positive Xpert MTB/RIF results (0·39% of those screened and 0·75% of those with valid sputum results). The positive predictive value of Xpert MTB/RIF was 61·0% (95% CI 52·8-68·7) when compared against a positive sputum culture and 83·9% (76·8-89·2) when compared against a positive sputum culture or chest radiograph consistent with active tuberculosis. On the basis of these positive predictive values, the specificity of Xpert MTB/RIF was determined to be between 99·78% (95% CI 99·71-99·84) and 99·93% (99·88-99·96). The positive predictive value and specificity of Xpert MTB/RIF in the context of community-wide screening for tuberculosis is substantially higher than that predicted in previous studies. Our findings support the potential role of Xpert MTB/RIF as a primary screening tool to detect prevalent cases of tuberculosis in the community. Australian National Health and Medical Research Council. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel sputum transport solution eliminates cold chain and supports routine tuberculosis testing in Nepal.

PubMed

Maharjan, Bhagwan; Shrestha, Bhabana; Weirich, Alexandra; Stewart, Andrew; Kelly-Cirino, Cassandra D

2016-12-01

This preliminary study evaluated the transport reagent OMNIgene SPUTUM (OMS) in a real-world, resource-limited setting: a zonal hospital and national tuberculosis (TB) reference laboratory, Nepal. The objectives were to: (1) assess the performance of OMS for transporting sputum from peripheral sites without cold chain stabilization; and (2) compare with Nepal's standard of care (SOC) for Mycobacterium tuberculosis smear and culture diagnostics. Sixty sputa were manually split into a SOC sample (airline-couriered to the laboratory, conventional processing) and an OMS sample (OMS added at collection, no cold chain transport or processing). Smear microscopy and solid culture were performed. Transport was 0-8days. Forty-one samples (68%) were smear-positive using both methods. Of the OMS cultures, 37 (62%) were positive, 22 (36%) were negative, and one (2%) was contaminated. Corresponding SOC results were 32 (53%), 21 (35%), and seven (12%). OMS "rescued" six (i.e., missed using SOC) compared with one rescue using SOC. Of smear-positives, six SOC samples produced contaminated cultures whereas only one OMS sample was contaminated. OMS reduced culture contamination from 12% to 2%, and improved TB detection by 9%. The results suggest that OMS could perform well as a no cold chain, long-term transport solution for smear and culture testing. The findings provide a basis for larger feasibility studies. Copyright © 2016 Ministry of Health, Saudi Arabia. Published by Elsevier Ltd. All rights reserved.

Sputum Microscopy With Fluorescein Diacetate Predicts Tuberculosis Infectiousness

PubMed Central

Datta, Sumona; Sherman, Jonathan M; Tovar, Marco A; Bravard, Marjory A; Valencia, Teresa; Montoya, Rosario; Quino, Willi; D’Arcy, Nikki; Ramos, Eric S; Gilman, Robert H; Evans, Carlton A

2017-01-01

Abstract Background Sputum from patients with tuberculosis contains subpopulations of metabolically active and inactive Mycobacterium tuberculosis with unknown implications for infectiousness. Methods We assessed sputum microscopy with fluorescein diacetate (FDA, evaluating M. tuberculosis metabolic activity) for predicting infectiousness. Mycobacterium tuberculosis was quantified in pretreatment sputum of patients with pulmonary tuberculosis using FDA microscopy, culture, and acid-fast microscopy. These 35 patients’ 209 household contacts were followed with prevalence surveys for tuberculosis disease for 6 years. Results FDA microscopy was positive for a median of 119 (interquartile range [IQR], 47–386) bacteria/µL sputum, which was 5.1% (IQR, 2.4%–11%) the concentration of acid-fast microscopy–positive bacteria (2069 [IQR, 1358–3734] bacteria/μL). Tuberculosis was diagnosed during follow-up in 6.4% (13/209) of contacts. For patients with lower than median concentration of FDA microscopy–positive M. tuberculosis, 10% of their contacts developed tuberculosis. This was significantly more than 2.7% of the contacts of patients with higher than median FDA microscopy results (crude hazard ratio [HR], 3.8; P = .03). This association maintained statistical significance after adjusting for disease severity, chemoprophylaxis, drug resistance, and social determinants (adjusted HR, 3.9; P = .02). Conclusions Mycobacterium tuberculosis that was FDA microscopy negative was paradoxically associated with greater infectiousness. FDA microscopy–negative bacteria in these pretreatment samples may be a nonstaining, slowly metabolizing phenotype better adapted to airborne transmission. PMID:28510693

Invasive forms of Candida and Aspergillus in sputum samples of pulmonary tuberculosis patients attending the tuberculosis reference laboratory in Ghaemshahr, Northern Iran: An analysis of samples collected during the past 10years.

PubMed

Jabbari Amiri, Mohammad Reza; Aghili, Seyed Reza; Shokohi, Tahereh; Hedayati, Mohammad Taghi; Abastabar, Mahdi; Aliyali, Masoud; Jabbari Amiri, Masoumeh; Hasanpour, Hamid

2016-12-01

Tuberculosis (TB) continues to be a major public health problem worldwide. This is especially true in Northern Iran, which has high TB prevalence. The chronic nature of this disease is further exacerbated if it is accompanied by fungal infection, which usually remains undiagnosed and thus untreated. Thus, mycotic infections add fatal dimensions to pulmonary TB. Our objective was to determine the prevalence of invasive forms of fungal elements in sputum samples collected from patients with pulmonary TB at a reference laboratory in Ghaemshahr, Northern Iran, during the past 10years. In this retrospective study, sputum samples collected from 430 patients were examined. Pulmonary TB in patients was confirmed in our laboratory, and samples obtained during the period from March 2006 to February 2016 were analyzed. The sputum samples were subjected to biological (bacterial) staining (Ziehl-Neelsen and fluorochrome) and mycological investigation using KOH + Calcofluor White (Sigma-Aldrich, India) by fluorescent microscopy and fungal culture on Sabouraud dextrose agar (Sigma-Aldrich, India) and CHROMagar (Paris, France). Invasive forms of fungal pathogens were observed as co-infection with Mycobacterium tuberculosis in 28/430 cases (6.51%). The frequency of Aspergillus, both branching and dichotomous infection, accounted for 3.72% (16/430): Aspergillus flavus, 1.63%; Aspergillus fumigatus, 1.16%; Aspergillus niger, 0.69%; and Aspergillus oryzae, 0.23%, respectively. Blastoconidia and pseudohyphae forms of yeast were observed as co-infection with M. tuberculosis in 2.79% (12/430) of the cases: Candida albicans, 1.86%; Candida krusei, 0.46%; and other Candida species, 0.46%, respectively. Northern Iran is a critical region in the TB world and multidrug-resistant TB is a serious problem in this region. Although it is believed that there exists a commensal relationship between fungus and TB infections, the invasive forms of fungal pathogens and their co-infection can be caused by increasing disability and failure of treatment. Diagnosis of secondary or co-existing fungal infections in TB is most important for reducing the mortality and morbidity of these patients. Copyright © 2016.

Diagnostic utility of a line probe assay for multidrug resistant-TB in smear-negative pulmonary tuberculosis.

PubMed

Singh, Binit Kumar; Sharma, Surendra K; Sharma, Rohini; Sreenivas, Vishnubhatla; Myneedu, Vithal P; Kohli, Mikashmi; Bhasin, Dinkar; Sarin, Sanjay

2017-01-01

To evaluate the performance of Genotype MTBDRplus VER 2.0 in the diagnosis of Mycobacterium tuberculosis (MTB) in sputum smear-negative pulmonary TB cases. A total of 572 Ziehl-Neelsen sputum smear-negative samples were selected and subjected to line probe assay (Genotype MTBDRplus VER 2.0), and culture in mycobacterial growth indicator tube (MGIT-960). Immunochromatographic test was used to confirm the MTB-complex (MTBC) in culture-positive samples and phenotypic drug-susceptibility testing was done using MGIT-960. The line probe assay was able to diagnose MTBC in 38.2% (213/558) of specimens after excluding 14 nontuberculous mycobacteria. Sensitivity and specificity of the assay were 68.4% and 89.3% respectively, considering MGIT-960 culture as gold standard after excluding contaminated and invalid results. On comparing with composite reference standard, the assay had 71.5% sensitivity and 100% specificity in the diagnosis of tuberculosis. The sensitivity and specificity for detecting resistance to rifampicin (RMP) were 100% and 99.24% respectively and for resistance to isoniazid (INH) were 97.62% and 98.55%, respectively. Genotype MTBDRplus VER 2.0 is a rapid and precise diagnostic tool for detection of MTB in sputum smear-negative samples. It also facilitates accurate diagnosis of RMP and INH resistance within turn around-time.

Performance of the Abbott RealTime MTB RIF/INH resistance assay when used to test Mycobacterium tuberculosis specimens from Bangladesh.

PubMed

Kostera, Joshua; Leckie, Gregor; Abravaya, Klara; Wang, Hong

2018-01-01

The Abbott RealTime MTB RIF/INH Resistance Assay (RT MTB RIF/INH) is an assay for the detection of rifampicin (RIF)- and/or isoniazid (INH)-resistant Mycobacterium tuberculosis (MTB). The assay can be used to test sputum, bronchial alveolar lavage, and N-Acetyl-L-Cysteine (NALC)/NaOH pellets prepared from these samples. The assay can be used in direct testing mode, or in reflex mode following a MTB positive result produced by its companion assay, Abbott RT MTB. In this study, the direct testing mode was used to test paired sputum and NALC/NaOH pellets prepared from sputum collected from Bangladesh TB patients. One hundred and thirty two paired samples were tested. The RT MTB RIF/INH inhibition rate was 0%. One hundred and twenty-two paired samples had results above the assay limit of detection and were analyzed by comparing with results from phenotypic drug sensitivity testing, GeneXpert MTB/RIF (Xpert), and MTBDR plus (Hain). RT MTB RIF/INH results were in good agreement with those of GeneXpert and Hain. The ability of this assay to detect RIF and INH resistance may contribute to the global control of multidrug resistant tuberculosis.

Carbon nanotube and nanofiber exposure and sputum and blood biomarkers of early effect among U.S. workers.

PubMed

Beard, John D; Erdely, Aaron; Dahm, Matthew M; de Perio, Marie A; Birch, M Eileen; Evans, Douglas E; Fernback, Joseph E; Eye, Tracy; Kodali, Vamsi; Mercer, Robert R; Bertke, Stephen J; Schubauer-Berigan, Mary K

2018-07-01

Carbon nanotubes and nanofibers (CNT/F) are increasingly used for diverse applications. Although animal studies suggest CNT/F exposure may cause deleterious health effects, human epidemiological studies have typically been small, confined to single workplaces, and limited in exposure assessment. We conducted an industrywide cross-sectional epidemiological study of 108 workers from 12 U.S. sites to evaluate associations between occupational CNT/F exposure and sputum and blood biomarkers of early effect. We assessed CNT/F exposure via personal breathing zone, filter-based air sampling to measure background-corrected elemental carbon (EC) (a CNT/F marker) mass and microscopy-based CNT/F structure count concentrations. We measured 36 sputum and 37 blood biomarkers. We used factor analyses with varimax rotation to derive factors among sputum and blood biomarkers separately. We used linear, Tobit, and unconditional logistic regression models to adjust for potential confounders and evaluate associations between CNT/F exposure and individual biomarkers and derived factors. We derived three sputum and nine blood biomarker factors that explained 78% and 67%, respectively, of the variation. After adjusting for potential confounders, inhalable EC and total inhalable CNT/F structures were associated with the most sputum and blood biomarkers, respectively. Biomarkers associated with at least three CNT/F metrics were 72 kDa type IV collagenase/matrix metalloproteinase-2 (MMP-2), interleukin-18, glutathione peroxidase (GPx), myeloperoxidase, and superoxide dismutase (SOD) in sputum and MMP-2, matrix metalloproteinase-9, metalloproteinase inhibitor 1/tissue inhibitor of metalloproteinases 1, 8-hydroxy-2'-deoxyguanosine, GPx, SOD, endothelin-1, fibrinogen, intercellular adhesion molecule 1, vascular cell adhesion protein 1, and von Willebrand factor in blood, although directions of associations were not always as expected. Inhalable rather than respirable CNT/F was more consistently associated with fibrosis, inflammation, oxidative stress, and cardiovascular biomarkers. Published by Elsevier Ltd.

Lack of association between increased mitochondrial DNA4977 deletion and ATP levels of sputum cells from chronic obstructive pulmonary disease patients versus healthy smokers.

PubMed

Karimova, A; Oltulu, Y M; Azaklı, H; Kara, M; Ustek, D; Tutluoglu, B; Onaran, I

2017-05-01

In this study we looked at smokers with and without chronic obstructive pulmonary disease (COPD) patients in order to evaluate the incidence of 4977 base pair (bp) mtDNA (mtDNA 4977 ) deletion and mtDNA copy number in sputum cells and in peripheral blood leukocytes (PBLs) in relation to mitochondrial function and oxidative stress status. Twenty-five COPD patients who were current smokers, 22 smokers and 23 healthy nonsmokers (for only PBLs studies) participated in this study. The 4977-bp deletion was detected in all examined samples within 40 cyles of PCR amplification, using a quantitative real time PCR. The frequency of the mtDNA 4977 was significantly higher in the sputum cells of patients with COPD compared to smokers without COPD (p < 0.0001). This difference was not observed in PBLs. Levels of cellular oxidative stress were significantly higher in the sputum cells of subjects with COPD than in the smoker group. However, mtDNA copy number, mitochondrial membrane potential (ΔΨm) and cellular ATP levels in PBLs and sputum cells were not significantly different between the studied groups. The Pearson analysis revealed no correlations between the accumulation of mtDNA 4977 , and intracellular ATP content and ΔΨm values of the sputum cells, although there was a positive correlation between the increase in the percentage of deleted mtDNA 4977 and the levels of cellular oxidative stress in COPD patients (r = 0.80, p < 0.0001). Our studies may suggest that the accumulation of mtDNA 4977 in the sputum cells of smokers with COPD does not seem to have an important impact on mitochondrial dysfunction in relation to ATP production and ΔΨm when compared to those of healthy smokers.

Active case-finding for tuberculosis by mobile teams in Myanmar: yield and treatment outcomes.

PubMed

Myint, Ohnmar; Saw, Saw; Isaakidis, Petros; Khogali, Mohammed; Reid, Anthony; Hoa, Nguyen Binh; Kyaw, Thi Thi; Zaw, Ko Ko; Khaing, Tin Mi Mi; Aung, Si Thu

2017-06-02

Since 2005, the Myanmar National Tuberculosis Programme (NTP) has been implementing active case finding (ACF) activities involving mobile teams in hard-to-reach areas. This study revealed the contribution of mobile team activities to total tuberculosis (TB) case detection, characteristics of TB patients detected by mobile teams and their treatment outcomes. This was a descriptive study using routine programme data between October 2014 and December 2014. Mobile team activities were a one-stop service and included portable digital chest radiography (CXR) and microscopy of two sputum samples. The algorithm of the case detection included screening patients by symptoms, then by CXR followed by sputum microscopy for confirmation. Diagnosed patients were started on treatment and followed until a final outcome was ascertained. A total of 9 349 people with symptoms suggestive of TB were screened by CXR, with an uptake of 96.6%. Of those who were meant to undergo sputum smear microscopy, 51.4% had sputum examinations. Finally, 504 TB patients were identified by the mobile teams and the overall contribution to total TB case detection in the respective townships was 25.3%. Among total cases examined by microscopy, 6.4% were sputum smear positive TB. Treatment success rate was high as 91.8% in study townships compared to national rate 85% (2014 cohort). This study confirmed the feasibility and acceptability of ACF by mobile teams in hard-to-reach contexts, especially when equipped with portable, digital CXR machines that provided immediate results. However, the follow-up process of sputum examination created a significant barrier to confirmation of the diagnosis. In order to optimize the ACF through mobile team activity, future ACF activities were needed to be strengthened one stop service including molecular diagnostics or provision of sputum cups to all presumptive TB cases prior to CXR and testing if CXR suggestive of TB.

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis

PubMed Central

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X.C.; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-01-01

Abstract The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection. To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10. The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples. The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%. The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. PMID:29390519

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis.

PubMed

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X C; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-12-01

The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection.To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10.The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples.The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%.The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

Combination Treatment with High-Dose Vitamin C and Alpha-Tocopherol does not Enhance Respiratory-Tract Lining Fluid Vitamin C Levels in Asthmatics

PubMed Central

Hernandez, Michelle; Zhou, Haibo; Zhou, Bingqing; Robinette, Carole; Crissman, Kay; Hatch, Gary; Alexis, Neil E; Peden, David

2011-01-01

Oxidative stress plays a significant role in allergic airway inflammation. Supplementation with alpha-tocopherol (alone or combined with ascorbate/vitamin C) has been assessed as an intervention for allergic airway diseases with conflicting results. Enhancing levels of airway antioxidants with oral supplements has been suggested as an intervention to protect individuals from the effect of inhaled oxidants, although it is unclear whether supplementation changes tocopherol or vitamin C levels in both serum and airway fluids. Our objective was to obtain pilot safety and dosing data from 14 allergic asthmatic volunteers examining the effect of daily combination oral therapy with 500 mg alpha-tocopherol (αT) and 2 g vitamin C for 12 wk. We examined serum and airway fluid and cellular levels of alpha- and gamma-tocopherol (γT) and vitamin C to plan for future studies of these agents in asthma and allergic rhinitis. Six volunteers completed 12 wk of active treatment with αT and vitamin C and 8 completed placebo. Blood and sputum samples were obtained at baseline and at 6 wk and 12 wk of therapy and were analyzed for αT, γT, and vitamin C levels in the serum, sputum supernatant, and sputum cells. Combination treatment increased serum vitamin C and significantly decreased sputum αT and serum γT levels. No changes were found in sputum supernatant or sputum cell vitamin C or serum αT levels in the active treatment group. In conclusion, supplementation with αT and high-dose vitamin C does not augment vitamin C levels in the respiratory-tract lining fluid. PMID:18932058

Can inflammatory markers in induced sputum be used to detect phenotypes and endotypes of pediatric severe therapy-resistant asthma?

PubMed

Eller, Miriam C N; Vergani, Karina P; Saraiva-Romanholo, Beatriz M; Antonangelo, Leila; Leone, Claudio; Rodrigues, Joaquim C

2018-06-05

The phenotypes and endotypes of severe therapy-resistant asthma (STRA) have not been fully elucidated in children. The aim of the present study was to investigate inflammatory markers in the induced sputum of children with STRA and to compare them with those present in a group of children who achieved control. A prospective cohort of children (6-18 years of age) diagnosed with severe asthma (GINA criteria) who had undergone treatment for at least 6 months was comprehensively followed for 3 months. Inhalation technique, adherence to treatment, ACT score, and main comorbidities were assessed. Induced sputum samples were collected for cytology analysis and quantitative assessment of cytokines; the participants also underwent spirometry, plethysmography, and fractional exhaled nitric oxide (FeNO) measurement. Forty patients were included (average age 12.8 years; 62.5% male); of these, 13 (32.5%) were classified as STRA at the end of follow-up. There were no significant differences between the STRA and control groups in demographic data, functional test results, or FeNO levels. The eosinophilic inflammatory pattern predominated in both groups; however, the STRA group showed a proportionally higher percentage of sputum neutrophils (P < 0.05). The median sputum levels of the cytokines IL-10, GM-CSF, IFN-γ, and TNF-α were significantly higher in the STRA group (P < 0.05). GM-CSF and TNF-α levels showed inverse correlations with ACT scores. The presence of neutrophils, the cytokines IL-10, and IFN-γ and, more particularly, TNF-α, and GM-CSF in the sputum may play an important role in the pathophysiological mechanism of STRA in children and adolescents. Specific antagonists for these cytokines may represent a future therapeutic strategy. © 2018 Wiley Periodicals, Inc.

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

PubMed

van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

2017-10-01

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Contaminant concentration in environmental samples using LIBS and CF-LIBS

NASA Astrophysics Data System (ADS)

Pandhija, S.; Rai, N. K.; Rai, A. K.; Thakur, S. N.

2010-01-01

The present paper deals with the detection and quantification of toxic heavy metals like Cd, Co, Pb, Zn, Cr, etc. in environmental samples by using the technique of laser-induced breakdown spectroscopy (LIBS) and calibration-free LIBS (CF-LIBS). A MATLABTM program has been developed based on the CF-LIBS algorithm given by earlier workers and concentrations of pollutants present in industrial area soil have been determined. LIBS spectra of a number of certified reference soil samples with varying concentrations of toxic elements (Cd, Zn) have been recorded to obtain calibration curves. The concentrations of Cd and Zn in soil samples from the Jajmau area, Kanpur (India) have been determined by using these calibration curves and also by the CF-LIBS approach. Our results clearly demonstrate that the combination of LIBS and CF-LIBS is very useful for the study of pollutants in the environment. Some of the results have also been found to be in good agreement with those of ICP-OES.

Anti-inflammatory effects of tobramycin and a copper–tobramycin complex with superoxide dismutase-like activity

PubMed Central

Gziut, M; MacGregor, HJ; Nevell, TG; Mason, T; Laight, D; Shute, JK

2013-01-01

Background and Purpose Airway inflammation in cystic fibrosis (CF) patients is characterized by accumulations of neutrophils in the airway and T cells in bronchial tissue, with activation of platelets in the circulation. CF patients are routinely treated with systemic or inhaled tobramycin for airway infection with Pseudomonas aeruginosa. Clinical trials have indicated an anti-inflammatory effect of tobramycin beyond its bactericidal activity. Here, we investigate the anti-inflammatory properties of tobramycin in vitro and consider if these relate to the ability of tobramycin to bind copper, which is elevated in blood and sputum in CF. Experimental Approach A copper–tobramycin complex was synthesized. The effect of tobramycin and copper–tobramycin on neutrophil activation and migration of T cells and neutrophils across human lung microvascular endothelial cells in response to thrombin-activated platelets were investigated in vitro. Tobramycin uptake was detected by immunocytochemistry. Intracellular reactive oxygen species were detected using the fluorescent indicator, 2′,7′-dichlorofluorescein diacetate (DCFDA). Neutrophil superoxide, hydrogen peroxide and neutrophil elastase activity were measured using specific substrates. Copper was measured using atomic absorption spectroscopy. Key Results Tobramycin and copper–tobramycin were taken up by endothelial cells via a heparan sulphate-dependent mechanism and significantly inhibited T-cell and neutrophil transendothelial migration respectively. Copper–tobramycin has intracellular and extracellular superoxide dismutase-like activity. Neutrophil elastase inhibition by α1-antitrypsin is enhanced in the presence of copper–tobramycin. Tobramycin and copper–tobramycin are equally effective anti-pseudomonal antibiotics. Conclusions and Implications Anti-inflammatory effects of tobramycin in vivo may relate to the spontaneous formation of a copper–tobramycin complex, implying that copper–tobramycin may be more effective therapy. PMID:23072509

Ciprofloxacin DPI: a randomised, placebo-controlled, phase IIb efficacy and safety study on cystic fibrosis.

PubMed

Dorkin, Henry L; Staab, Doris; Operschall, Elisabeth; Alder, Jeff; Criollo, Margarita

2015-01-01

Treatment of infective bronchitis involving Pseudomonas aeruginosa is a cornerstone of care in patients with cystic fibrosis (CF). This phase IIb, randomised, double-blind, placebo-controlled study assessed the efficacy and safety of ciprofloxacin dry powder for inhalation (DPI) in this population. Patients with CF, ≥12 years of age (N=286), were randomised to ciprofloxacin DPI (32.5 mg (n=93) or 48.75 mg (n=93)), or corresponding placebo (32.5 mg, n=65; 48.75 mg, n=35) twice daily for 28 days. The primary objective was the change in forced expiratory volume in 1 s (FEV1) from baseline (day 0) to end of treatment (day 29) in the intent-to-treat population for ciprofloxacin DPI compared with the corresponding placebo group. The primary effectiveness objective was not met; there were no significant differences in change in FEV1 between ciprofloxacin DPI and the corresponding placebo group for either dose (p=0.154). However, in pooled analyses, FEV1 decline from baseline to treatment end was significantly lower with ciprofloxacin DPI than with placebo (pooled data; p=0.02). Ciprofloxacin DPI showed positive effects on sputum bacterial load and quality of life, but these effects were not maintained at the 4-week follow-up. Ciprofloxacin DPI was well tolerated and there were no significant differences in type/incidence of treatment-emergent adverse events by treatment group (p=0.115). Further investigations are needed to determine the full scope of the beneficial effects of ciprofloxacin DPI for patients with CF. Clinicaltrials.gov NCT00645788; EudraCT 2008-008314-40.

Ciprofloxacin DPI: a randomised, placebo-controlled, phase IIb efficacy and safety study on cystic fibrosis

PubMed Central

Dorkin, Henry L; Staab, Doris; Operschall, Elisabeth; Alder, Jeff; Criollo, Margarita

2015-01-01

Background Treatment of infective bronchitis involving Pseudomonas aeruginosa is a cornerstone of care in patients with cystic fibrosis (CF). This phase IIb, randomised, double-blind, placebo-controlled study assessed the efficacy and safety of ciprofloxacin dry powder for inhalation (DPI) in this population. Methods Patients with CF, ≥12 years of age (N=286), were randomised to ciprofloxacin DPI (32.5 mg (n=93) or 48.75 mg (n=93)), or corresponding placebo (32.5 mg, n=65; 48.75 mg, n=35) twice daily for 28 days. The primary objective was the change in forced expiratory volume in 1 s (FEV1) from baseline (day 0) to end of treatment (day 29) in the intent-to-treat population for ciprofloxacin DPI compared with the corresponding placebo group. Results The primary effectiveness objective was not met; there were no significant differences in change in FEV1 between ciprofloxacin DPI and the corresponding placebo group for either dose (p=0.154). However, in pooled analyses, FEV1 decline from baseline to treatment end was significantly lower with ciprofloxacin DPI than with placebo (pooled data; p=0.02). Ciprofloxacin DPI showed positive effects on sputum bacterial load and quality of life, but these effects were not maintained at the 4-week follow-up. Ciprofloxacin DPI was well tolerated and there were no significant differences in type/incidence of treatment-emergent adverse events by treatment group (p=0.115). Conclusions Further investigations are needed to determine the full scope of the beneficial effects of ciprofloxacin DPI for patients with CF. Trial registration number Clinicaltrials.gov NCT00645788; EudraCT 2008-008314-40. PMID:26688732

Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung

PubMed Central

Harmer, Christopher J.; Wynn, Matthew; Pinto, Rachel; Cordwell, Stuart; Rose, Barbara R.; Harbour, Colin; Triccas, James A.; Manos, Jim

2015-01-01

Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1. PMID:26252386

Aspergillus fumigatus chronic colonization and lung function decline in cystic fibrosis may have a two-way relationship.

PubMed

Noni, M; Katelari, A; Dimopoulos, G; Doudounakis, S-E; Tzoumaka-Bakoula, C; Spoulou, V

2015-11-01

Aspergillus fumigatus is commonly found in cystic fibrosis (CF) airways. Our aim was to assess the relationship between A. fumigatus chronic colonization and lung function in CF patients. A case-control study of CF patients born from 1989 to 2002 was performed. Medical records were reviewed from the time of initial diagnosis until December 2013. Chronic colonization was defined as two or more positive sputum cultures in a given year. Each patient chronically colonized with A. fumigatus was matched with three control patients (never colonized by A. fumigatus) for age, sex, and year of birth (±3 years). A number of parameters were recorded and analyzed prospectively. The primary outcome measure was the difference in forced expiratory volume in 1 s (FEV1) in percent predicted between groups. Linear mixed models were used for longitudinal analyses to evaluate the relationship between A. fumigatus chronic colonization and lung function during a 7-year period and study the lung function 4 years before the time of enrollment (t0). Twenty patients had chronic colonization and were matched with 60 controls. A significant difference in lung function was detected throughout the 7-year period after adjustment for confounders (est = 8.66, p = 0.020). Four years before t0, FEV1 baseline was the only factor associated with the course of lung function (est = 0.64, p < 0.001) and was significantly different between groups (p = 0.001). In conclusion, a decreased FEV1 baseline appears to be a risk factor for chronic colonization by A. fumigatus, which, in turn, may cause a faster deterioration of lung function.

Inhaled, dual release liposomal ciprofloxacin in non-cystic fibrosis bronchiectasis (ORBIT-2): a randomised, double-blind, placebo-controlled trial

PubMed Central

Serisier, David J; Bilton, Diana; De Soyza, Anthony; Thompson, Philip J; Kolbe, John; Greville, Hugh W; Cipolla, David; Bruinenberg, Paul; Gonda, Igor

2013-01-01

Background The delivery of antipseudomonal antibiotics by inhalation to Pseudomonas aeruginosa-infected subjects with non-cystic fibrosis (CF) bronchiectasis is a logical extension of treatment strategies successfully developed in CF bronchiectasis. Dual release ciprofloxacin for inhalation (DRCFI) contains liposomal ciprofloxacin, formulated to optimise airway antibiotic delivery. Methods Phase II, 24-week Australian/New Zealand multicentre, randomised, double-blind, placebo-controlled trial in 42 adult bronchiectasis subjects with ≥2 pulmonary exacerbations in the prior 12 months and ciprofloxacin-sensitive P aeruginosa at screening. Subjects received DRCFI or placebo in three treatment cycles of 28 days on/28 days off. The primary outcome was change in sputum P aeruginosa bacterial density to the end of treatment cycle 1 (day 28), analysed by modified intention to treat (mITT). Key secondary outcomes included safety and time to first pulmonary exacerbation—after reaching the pulmonary exacerbation endpoint subjects discontinued study drug although remained in the study. Results DRCFI resulted in a mean (SD) 4.2 (3.7) log10 CFU/g reduction in P aeruginosa bacterial density at day 28 (vs −0.08 (3.8) with placebo, p=0.002). DRCFI treatment delayed time to first pulmonary exacerbation (median 134 vs 58 days, p=0.057 mITT, p=0.046 per protocol). DRCFI was well tolerated with a similar incidence of systemic adverse events to the placebo group, but fewer pulmonary adverse events. Conclusions Once-daily inhaled DRCFI demonstrated potent antipseudomonal microbiological efficacy in adults with non-CF bronchiectasis and ciprofloxacin-sensitive P aeruginosa. In this modest-sized phase II study, DRCFI was also well tolerated and delayed time to first pulmonary exacerbation in the per protocol population. PMID:23681906

Xpert MTB/RIF assay for diagnosis of pulmonary tuberculosis in sputum specimens in remote health care facility.

PubMed

Geleta, Dereje Assefa; Megerssa, Yoseph Cherinet; Gudeta, Adugna Negussie; Akalu, Gizachew Taddesse; Debele, Melaku Tesfaye; Tulu, Kassu Desta

2015-10-19

Xpert MTB/RIF assay is considered as a great advance over conventional smear and culture in the diagnosis of TB and MDR-TB by simultaneously detecting M.tuberculosis and rifampicin resistance bacilli. However, very little information regarding the performance characteristics of Xpert MTB/RIF assay is available in Ethiopia. Therefore, the purpose of this study was to evaluate the performance of Xpert MTB/RIF assay compared to conventional sputum smear and culture methods for the diagnosis of pulmonary tuberculosis in remote health care facility. A paired expectorated sputum samples were obtained from 227 consecutively recruited patients with signs and symptoms suggestive of tuberculosis at Karamara hospital during December 2013 to May 2014. One of the sputum specimen was tested directly by Ziehl-Neelsen staining and Xpert MTB/RIF assay without NALC-NaOH decontamination. The other of pair of sputa specimen was cultured for isolation of TB bacilli by conventional methods. Diagnostic performance of Xpert MTB/RIF assay and AFB smear microscopy were calculated against culture as the gold standard. Overall 25.5% (58/227) samples were positive for Mycobacterium tuberculosis complex (MTBC) by MGIT and/or LJ media of which 36.2% (21/58) and 65.5% (35/58) were positive by AFB smear microscopy and Xpert MTB/RIF respectively. The sensitivity, specificity, as well as the positive and negative predictive value of Xpert MTB/RIF assay were 65.5% (95% CI: 53.3-77.7%), 96.3% (95% CI: 93.4-99.2%), 86.4% (95% CI: 76.2-96.5%), and 88.6% (95% CI: 83.9-93.3%) respectively. Eighteen of 58 (31%) cases that were smear microscopy negative, were positive by Xpert MTB/RIF assay. Although Xpert MTB/RIF assay demonstrated high sensitivity in detecting MTBC in sputum specimens compared with conventional AFB smear microscopy, it demonstrated suboptimal sensitivity in smear negative patients compared to conventional culture.

Rapid Diagnosis of Smear-Negative Tuberculosis Using Immunology and Microbiology with Induced Sputum in HIV-Infected and Uninfected Individuals

PubMed Central

Breen, Ronan A. M.; Hardy, Gareth A. D.; Perrin, Felicity M. R.; Lear, Sara; Kinloch, Sabine; Smith, Colette J.; Cropley, Ian; Janossy, George; Lipman, Marc C. I.

2007-01-01

Rationale and Objectives Blood-based studies have demonstrated the potential of immunological assays to detect tuberculosis. However lung fluid sampling may prove superior as it enables simultaneous microbiological detection of mycobacteria to be performed. Until now this has only been possible using the expensive and invasive technique of broncho-alveolar lavage. We sought to evaluate an immunoassay using non-invasive induced-sputum to diagnose active tuberculosis. Methods and Results Prospective cohort study of forty-two spontaneous sputum smear-negative or sputum non-producing adults under investigation for tuberculosis. CD4 lymphocytes specific to purified-protein-derivative of Mycobacterium tuberculosis actively synthesising interferon-gamma were measured by flow cytometry and final diagnosis compared to immunoassay using a cut-off of 0.5%. Sixteen subjects (38%) were HIV-infected (median CD4 count [range] = 332 cells/µl [103–748]). Thirty-eight (90%) were BCG-vaccinated. In 27 subjects diagnosed with active tuberculosis, the median [range] percentage of interferon-gamma synthetic CD4+ lymphocytes was 2.77% [0–23.93%] versus 0% [0–2.10%] in 15 negative for active infection (p<0.0001). Sensitivity and specificity of the immunoassay versus final diagnosis of active tuberculosis were 89% (24 of 27) and 80% (12 of 15) respectively. The 3 positive assays in the latter group occurred in subjects diagnosed with quiescent/latent tuberculosis. Assay performance was unaffected by HIV-status, BCG-vaccination or disease site. Combining this approach with traditional microbiological methods increased the diagnostic yield to 93% (25 of 27) alongside acid-fast bacilli smear and 96% (26 of 27) alongside tuberculosis culture. Conclusions These data demonstrate for the first time that a rapid immunological assay to diagnose active tuberculosis can be performed successfully in combination with microbiological methods on a single induced-sputum sample. PMID:18092001

The secretory IgA system of lung secretions in chronic obstructive bronchitis: comparison of sputum with secretions obtained during fibreoptic bronchoscopy.

PubMed Central

Wiggins, J; Hill, S L; Stockley, R A

1984-01-01

The constituents of the secretory immunoglobulin A system (dimeric IgA, total secretory component and free secretory component) were measured in sputum sol phase, tracheal aspirates, and bronchoalveolar lavage fluids from 15 patients undergoing fibreoptic bronchoscopy. All of the proteins showed a progressive decrease in concentration from sputum to the bronchoalveolar lavage fluids (2p less than 0.001). Standardisation of samples by means of protein concentration ratios showed that all secretions were generally similar in respect of their secretory IgA profiles, although major differences remained in some individual patients. The between patient variability of the results was generally reduced by the use of protein concentration ratios, allowing closer comparison between subjects. When the secretion albumin concentration was used as a standard, however, it increased the variability of the sputum sol phase IgA components (2p less than 0.01), whereas it decreased the variability of the IgA components in the bronchoalveolar lavage fluid (2p less than 0.05). The role of albumin as a standard protein for assessing the secretory IgA system in lung secretions remains uncertain. PMID:6463931

Detection of lipoarabinomannan as a diagnostic test for tuberculosis.

PubMed Central

Sada, E; Aguilar, D; Torres, M; Herrera, T

1992-01-01

A coagglutination technique was established for the detection of lipoarabinomannan of Mycobacterium tuberculosis in human serum samples and evaluated for its utility in the diagnosis of tuberculosis at the Instituto Nacional de Enfermedades Respiratorias in Mexico City. The test had a sensitivity of 88% in patients with sputum-smear-positive active pulmonary tuberculosis. The sensitivity in patients with active pulmonary tuberculosis negative for acid-fast bacilli in sputum was 67%. Less favorable results were obtained for patients with AIDS and tuberculosis, with a sensitivity of 57%. The specificity in control patients with lung diseases different from tuberculosis and in healthy subjects was 100%. The positive predictive value was 100%, and the negative predictive value for patients with sputum-positive active pulmonary tuberculosis was 97%. The results of this study suggest that the detection of lipoarabinomannan is an accurate test for the diagnosis of pulmonary tuberculosis. PMID:1401008

Improvement of the fracture toughness of hydroxyapatite (HAp) by incorporation of carboxyl functionalized single walled carbon nanotubes (CfSWCNTs) and nylon

DOE PAGES

Khanal, Suraj P.; Mahfuz, Hassan; Rondinone, Adam Justin; ...

2015-11-12

The potential of improving the fracture toughness of synthetic hydroxyapatite (HAp) by incorporating carboxyl functionalized single walled carbon nanotubes (CfSWCNTs) and polymerized ε-caprolactam (nylon) was researched. A series of HAp samples with CfSWCNTs concentrations varying from 0 to 1.5 wt.%, without, and with nylon addition was prepared. X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) were used to characterize the samples. The three point bending test was applied to measure the fracture toughness of the composites. A reproducible value of 3.6 ± 0.3 MPa.√m was found for samples containing 1 wt.% CfSWCNTs and nylon. This valuemore » is in the range of the cortical bone fracture toughness. Lastly, the increase of the CfSWCNTs content results to decrease of the fracture toughness, and formation of secondary phases.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heimburger, D.C.; Alexander, C.B.; Birch, R.

To test whether changes in folate and vitamin B/sub 12/ nutrition modify the severity of potentially premalignant lesions identified by cytology in sputum samples of smokers, the authors conducted a randomized, controlled prospective intervention trial in smokers with bronchial squamous metaplasia. Seventy-three men with a history of 20 or more pack-years of cigarette smoking who had metaplasia on one or more sputum samples were stratified according to smoking level and randomly assigned to four months' treatment with either placebo or 10 mg of folate plus 500 ..mu..g of hydroxocobalamin. Direct cytological comparison of the two groups after four months showedmore » significantly greater reduction of atypia in the supplemented group. This provides preliminary evidence that atypical bronchial squamous metaplasia may be reduced by supplementation with folate and vitamin B/sub 12/. However, the significance of these findings is tempered by substantial spontaneous variation in sputum cytologies, the small study population, the short duration of the trial, and the supraphysiological doses of folate and B/sub 12/ used. The results should not be construed as pointing to a potential way of preventing lung cancer in individuals who continue to smoke or as supporting self-medication with large doses of folate or B/sub 12/ by smokers.« less

Peroxisome proliferator-activated receptor-γ in induced sputum is correlated with MMP-9/TIMP-1 imbalance and formation of emphysema in COPD patients.

PubMed

Zhou, Xiao-Ming; Hou, Gang; Gu, Dong-Xue; Wang, Qiu-Yue; Zhao, Li

2017-10-01

The development of chronic obstructive pulmonary disease (COPD) is modulated by the symmetry of matrix metalloproteinases (MMPs) and the counter-acting tissue inhibitors of metalloproteinases (TIMPs). We investigated the interaction between peroxisome proliferator-activated receptor gamma (PPARγ) expression and the imbalance of MMP-9/TIMP-1 in the induced sputum of stable COPD patients. Sixty-six stable COPD patients were enrolled and the induced sputum samples were gathered. The correlation between PPARγ and other index, including MMP-9, TIMP-1, pulmonary function and the index of emphysema-the percentage of low attenuation area (LAA%), was analyzed. PPARγ and TIMP-1 concentrations were decreased and the concentration of MMP-9 and the ratio of MMP9/TIMP1 were enhanced in the induced sputum of COPD patients, compared to the healthy controls. Among COPD patients, those with worse lung function or patients with emphysema exhibited increased MMP-9 expression with decreased TIMP-1 and PPARγ expression. Besides, the concentration of PPARγ of the induced sputum was correlated with the forced expiratory volume in one second percentage (FEV1%) positively and the expression of TIMP-1; while it was negatively correlated with the residual volume (RV), RV/total lung capacity (TLC), LAA%, and MMP-9 expression. Our findings reveal the protective role of PPARγ in the maintenance of the dynamic balance of MMP-9/TIMP-1 in COPD, thus providing evidence on which to base the potential COPD treatment.

Sputum Microscopy With Fluorescein Diacetate Predicts Tuberculosis Infectiousness.

PubMed

Datta, Sumona; Sherman, Jonathan M; Tovar, Marco A; Bravard, Marjory A; Valencia, Teresa; Montoya, Rosario; Quino, Willi; D'Arcy, Nikki; Ramos, Eric S; Gilman, Robert H; Evans, Carlton A

2017-09-01

Sputum from patients with tuberculosis contains subpopulations of metabolically active and inactive Mycobacterium tuberculosis with unknown implications for infectiousness. We assessed sputum microscopy with fluorescein diacetate (FDA, evaluating M. tuberculosis metabolic activity) for predicting infectiousness. Mycobacterium tuberculosis was quantified in pretreatment sputum of patients with pulmonary tuberculosis using FDA microscopy, culture, and acid-fast microscopy. These 35 patients' 209 household contacts were followed with prevalence surveys for tuberculosis disease for 6 years. FDA microscopy was positive for a median of 119 (interquartile range [IQR], 47-386) bacteria/µL sputum, which was 5.1% (IQR, 2.4%-11%) the concentration of acid-fast microscopy-positive bacteria (2069 [IQR, 1358-3734] bacteria/μL). Tuberculosis was diagnosed during follow-up in 6.4% (13/209) of contacts. For patients with lower than median concentration of FDA microscopy-positive M. tuberculosis, 10% of their contacts developed tuberculosis. This was significantly more than 2.7% of the contacts of patients with higher than median FDA microscopy results (crude hazard ratio [HR], 3.8; P = .03). This association maintained statistical significance after adjusting for disease severity, chemoprophylaxis, drug resistance, and social determinants (adjusted HR, 3.9; P = .02). Mycobacterium tuberculosis that was FDA microscopy negative was paradoxically associated with greater infectiousness. FDA microscopy-negative bacteria in these pretreatment samples may be a nonstaining, slowly metabolizing phenotype better adapted to airborne transmission. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

PubMed

Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

2017-11-06

Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

Effect of Treated Coconut Shell and Fiber on the Resilient Modulus of Double-layer Porous Asphalt at Different Aging

NASA Astrophysics Data System (ADS)

Ting, T. L.; Ramadhansyah, P. J.; Norhidayah, A. H.; Yaacob, H.; Hainin, M. R.; Ibrahim, M. H. Wan; Jayanti, D. S.; Abdullahi, A. M.

2018-04-01

Coconut shell (CS) and coconut fiber (CF) are new waste products that have been of growing interest recently in the highway asphalt pavement industry. This study investigated the effect of CS and CF on the resilient modulus of double-layer porous asphalt (DLPA). CS aggregate 5 mm in size was substituted for the DLPA at 5%, 10%, and 15% by weight, while CF was added to the asphalt at 0.3% and 0.5% by weight. Before mixing with other aggregates, the CS and CF were treated with 5%wt Sodium hydroxide (NaOH) to reduce their water absorption ability. The samples were prepared via the Marshall method. The result shows that DLPA with 10% CS aggregate has better resilient modulus under 25 °C for unaged and aged samples compared with the other substitution percentages. However, the sample with CF has a lower resilient modulus because the amount of CF has increased. In general, the substitution of 10% CS provided better resilient modulus among the other percentages.

Community acquired bacterial pneumonia: aetiology, laboratory detection and antibiotic susceptibility pattern.

PubMed

Akter, Sonia; Shamsuzzaman, S M; Jahan, Ferdush

2014-08-01

This cross sectional study was conducted to identify the common bacterial causes of community acquired pneumonia (CAP) from sputum and blood by culture and polymerase chain reaction (PCR) and to evaluate the effectiveness of these tests. A total of 105 sputum and blood samples were collected from patients with pneumonia on clinical suspicion. Common causative bacterial agents of pneumonia were detected by Gram staining, cultures, biochemical tests and PCR. Among 55 sputum culture positive cases, a majority (61.82%) of the patients were in the age group between 21-50 years and the ratio between male and female was 2.5:1. Most (61.90%) of the cases were from the lower socio-economic group. Out of 105 samples, 23 (37.12%) were positive by Gram stain, 29 (27.62%) yielded growth in culture media and 37 (35.24%) were positive by PCR for Streptococcus pneumoniae and Haemophilus influenzae. Streptococcus pneumoniae was the most common aetiological agent (19.05%) followed by Klebsiella pneumoniae (13.33%), Haemophilus influenzae (8.57%) and Pseudomonas aeruginosa (5.71%). Multiplex PCR is a useful technique for rapid diagnosis of bacterial causes of pneumonia directly from sputum and blood. Considering culture as a gold standard, the sensitivity of PCR was 96.55% and specificity was 88.15%. More than 80% of Streptococcus pneumoniae isolates were found to be sensitive to ampicillin, amoxycillinclavulanate, and ceftriaxone. Susceptibilities to other antimicrobials ranged from 65% for azithromycin to 70% for levofloxacin. On the other hand, the Gram negative organisms were more sensitive to meropenem, ceftriaxone, amoxycillin-clavulanate and amikacin.

Magic angle spinning nuclear magnetic resonance apparatus and process for high-resolution in situ investigations

DOEpatents

Hu, Jian Zhi; Sears, Jr., Jesse A.; Hoyt, David W.; Mehta, Hardeep S.; Peden, Charles H. F.

2015-11-24

A continuous-flow (CF) magic angle sample spinning (CF-MAS) NMR rotor and probe are described for investigating reaction dynamics, stable intermediates/transition states, and mechanisms of catalytic reactions in situ. The rotor includes a sample chamber of a flow-through design with a large sample volume that delivers a flow of reactants through a catalyst bed contained within the sample cell allowing in-situ investigations of reactants and products. Flow through the sample chamber improves diffusion of reactants and products through the catalyst. The large volume of the sample chamber enhances sensitivity permitting in situ .sup.13C CF-MAS studies at natural abundance.

Dating groundwater with trifluoromethyl sulfurpentafluoride (SF 5CF3), sulfur hexafluoride (SF6), CF 3Cl (CFC-13), and CF2Cl2 (CFC-12)

USGS Publications Warehouse

Busenberg, E.; Plummer, Niel

2008-01-01

[1] A new groundwater dating procedure using the transient atmospheric signal of the environmental tracers SF5CF3, CFC-13, SF6, and CFC-12 was developed. The analytical procedure determines concentrations of the four tracers in air and water samples. SF 5CF3 and CFC-13 can be used to date groundwaters in some environments where the CFCs and SF6 have previously failed because these new tracers have increasing atmospheric input functions, no known terrigenic source, and are believed to be stable under reducing conditions. SF5CF3 has a dating range from 1970 to modern; the mixing ratio (mole fraction) in North American air has increased from the detection limit of 0.005 parts per trillion (ppt) to the 2006 mole fraction of about 0.16 ppt. No evidence was found for degradation of SF5CF3 in laboratory anaerobic systems. The solubility of SF5CF3 was measured in water from 1 to 35??C. Groundwater samples that contained large amounts of terrigenic SF6 did not contain terrigenic SF 5CF3. CFC-13 is a trace atmospheric gas with a dating range in groundwater of about 1965 to modem. CFC-13 has been used primarily in very low-temperature refrigeration; thus groundwater environments are less likely to be contaminated with nonatmospheric sources as compared to other widely used CFCs. Because of the low solubility of SF5CF3 and CFC-13 in water, an excess air correction must be applied to the apparent ages. The new dating procedure was tested in water samples from wells and springs from Maryland, Virginia, and West Virginia.

New approach for cystic fibrosis diagnosis based on chloride/potassium ratio analyzed in non-invasively obtained skin-wipe sweat samples by capillary electrophoresis with contactless conductometric detection.

PubMed

Ďurč, Pavol; Foret, František; Pokojová, Eva; Homola, Lukáš; Skřičková, Jana; Herout, Vladimír; Dastych, Milan; Vinohradská, Hana; Kubáň, Petr

2017-05-01

A new approach for sweat analysis used in cystic fibrosis (CF) diagnosis is proposed. It consists of a noninvasive skin-wipe sampling followed by analysis of target ions using capillary electrophoresis with contactless conductometric detection (C4D). The skin-wipe sampling consists of wiping a defined skin area with precleaned cotton swab moistened with 100 μL deionized water. The skin-wipe sample is then extracted for 3 min into 400 μL deionized water, and the extract is analyzed directly. The developed sampling method is cheap, simple, fast, and painless, and can replace the conventional pilocarpine-induced sweat chloride test commonly applied in CF diagnosis. The aqueous extract of the skin-wipe sample content is analyzed simultaneously by capillary electrophoresis with contactless conductometric detection using a double opposite end injection. A 20 mmol/L L-histidine/2-(N-morpholino)ethanesulfonic acid and 2 mmol/L 18-crown-6 at pH 6 electrolyte can separate all the major ions in less than 7 min. Skin-wipe sample extracts from 30 study participants-ten adult patients with CF (25-50 years old), ten pediatric patients with CF (1-15 years old), and ten healthy control individuals (1-18 years old)-were obtained and analyzed. From the analyzed ions in all samples, a significant difference between chloride and potassium concentrations was found in the CF patients and healthy controls. We propose the use of the Cl - /K + ratio rather than the absolute Cl - concentration and a cutoff value of 4 in skin-wipe sample extracts as an alternative to the conventional sweat chloride analysis. The proposed Cl - /K + ion ratio proved to be a more reliable indicator, is independent of the patient's age, and allows better differentiation between non-CF individuals and CF patients having intermediate values on the Cl - sweat test. Figure New approach for cystic fibrosis diagnosis based on skin-wipe sampling of forearm and analysis of ionic content (Cl - /K + ratio) in skin-wipe extracts by capillary electrophoresis with contactless conductometric detection.

Skin Biomarkers for Cystic Fibrosis: A Potential Non-Invasive Approach for Patient Screening.

PubMed

Esteves, Cibele Zanardi; de Aguiar Dias, Letícia; de Oliveira Lima, Estela; de Oliveira, Diogo Noin; Rodrigues Melo, Carlos Fernando Odir; Delafiori, Jeany; Souza Gomez, Carla Cristina; Ribeiro, José Dirceu; Ribeiro, Antônio Fernando; Levy, Carlos Emílio; Catharino, Rodrigo Ramos

2017-01-01

Cystic fibrosis (CF) is a disabling genetic disease with an increased prevalence in European heritage populations. Currently, the most used technique for collection of CF samples and diagnosis is provided through uncomfortable tests, with uncertain results, mostly based on chloride concentration in sweat. Since CF mutation induces many metabolic changes in patients, exploring these alterations might be an alternative to visualize potential biomarkers that could be used as interesting tools for further diagnostic upgrade, prioritizing simplicity, low cost, and quickness. This contribution describes an accurate strategy to provide potential biomarkers related to CF, which may be understood as a potential tool for new diagnostic approaches and/or for monitoring disease evolution. Therefore, the present proposal consists of using skin imprints on silica plates as a way of sample collection, followed by direct-infusion high-resolution mass spectrometry and multivariate data analysis, intending to identify metabolic changes in skin composition of CF patients. Metabolomics analysis allowed identifying chemical markers that can be traced back to CF in patients' skin imprints, differently from control subjects. Seven chemical markers from several molecular classes were elected, represented by bile acids, a glutaric acid derivative, thyrotropin-releasing hormone, an inflammatory mediator, a phosphatidic acid, and diacylglycerol isomers, all reflecting metabolic disturbances that occur due to of CF. The comfortable method of sample collection combined with the identified set of biomarkers represent potential tools that open the range of possibilities to manage CF and follow the disease evolution. This exploratory approach points to new perspectives about the development of diagnostic assay using biomarkers and the management CF.

Prevalence of Paragonimus uterobilateralis infection in children in a Liberian village.

PubMed

Sachs, R; Albiez, E J; Voelker, J

1986-01-01

Stool and sputum samples from 127 children in an endemic paragonimiasis area in Liberia, West Africa, were examined for the presence of lung fluke eggs. Samples from nine children (7%) were positive. The eggs were identified as those of Paragonimus uterobilateralis.

Improving Diagnostic of Pulmonary Tuberculosis in HIV Patients by Bronchoscopy: A Cross Sectional Study.

PubMed

Santoso, Prayudi; Soeroto, Arto Y; Juniati, Rianita; Hartantri, Yovita; Wisaksana, Rudi; Alisjabana, Bachti; Nataprawira, Heda M; Parwati, Ida

2017-10-01

diagnostic of pulmonary TB in HIV patients is a problem due to non specific clinical features, or radiological appearance. HIV patients with CD4≤200 cells/mL infected with M. tuberculosis have less capacity in containing M. tuberculosis, developing granulomas, casseous necrosis, or cavities. This condition is caused by weakend inflammatory which later reduced sputum production and may cause false negative result. This study aimed to assess differences in the positivity level of acid fast bacilli (AFB) and cultures of M. tuberculosis from non-bronchoscopic sputum (spontaneous and induced sputum) compared to bronchoscopic sputum (bronchoalveolar lavage) in HIV positive patients suspected pulmonary tuberculosis with CD4<200 cells/μL. this cross sectional study was conducted in adult HIV patients treated in Hasan Sadikin Hospital with CD4≤200 cells/μL suspected with pulmonary tuberculosis by using paired comparative analytic test. All patients expelled sputum spontaneously or with sputum induction on the first day. On the next day, bronchoalveolar lavage (BAL) was performed. The two samples obtained from two methods were examined by AFB examination with staining Ziehl Neelsen (ZN) and cultured of M. tuberculosis on solid media Ogawa on all patients. Positivity, sensitivity and increased sensitivity of AFB and culture of M. tuberculosis in the non bronchoscopic and bronchoscopic groups were compared. there were differences in the positivity level of AFB with ZN staining between non-bronchoscopic and bronchoscopic groups which were 7/40 (17.5%) vs 20/40 (50.0%) (p<0.001). The differences between the cultures of non-bronchoscopic and bronchoscopic groups were 16/40 (40.0%) vs 23/40 (57.5%) (p=0.039). Bronchoscopic sputum increased the positivity level of the ZN AFB examination by 32.5% (from 17.5% to 50.0%) as well as on culture examination by 17.5% (from 40.0% to 57.5%). Bronchoalveolar lavage can improve the positivity level of smears and cultures in patients suspected of pulmonary TB in HIV patients with CD4<200 cells/μL.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saccomanno, G.

This work, supported by the United States Department of Energy, continues to add data on the health effects of cigarette smoking and radon exposure. Since the beginning of this contract, 473 sputum samples have been collected from 286 uranium workers who are routinely screened in an effort to identify cell changes that could signal possible progression to lung cancer; seven new lung cancer cases have been identified during this period. At this time, there are 426 lung cancer cases in the uranium miner tumor registry with diagnostic slides from surgery and/or autopsy; an additional 40 cases have been diagnosed withmore » sputum cytology only.« less

Sputum culture conversion as a prognostic marker for end-of-treatment outcome in patients with multidrug-resistant tuberculosis: a secondary analysis of data from two observational cohort studies.

PubMed

Kurbatova, Ekaterina V; Cegielski, J Peter; Lienhardt, Christian; Akksilp, Rattanawadee; Bayona, Jaime; Becerra, Mercedes C; Caoili, Janice; Contreras, Carmen; Dalton, Tracy; Danilovits, Manfred; Demikhova, Olga V; Ershova, Julia; Gammino, Victoria M; Gelmanova, Irina; Heilig, Charles M; Jou, Ruwen; Kazennyy, Boris; Keshavjee, Salmaan; Kim, Hee Jin; Kliiman, Kai; Kvasnovsky, Charlotte; Leimane, Vaira; Mitnick, Carole D; Quelapio, Imelda; Riekstina, Vija; Smith, Sarah E; Tupasi, Thelma; van der Walt, Martie; Vasilyeva, Irina A; Via, Laura E; Viiklepp, Piret; Volchenkov, Grigory; Walker, Allison Taylor; Wolfgang, Melanie; Yagui, Martin; Zignol, Matteo

2015-03-01

Sputum culture conversion is often used as an early microbiological endpoint in phase 2 clinical trials of tuberculosis treatment on the basis of its assumed predictive value for end-of-treatment outcome, particularly in patients with drug-susceptible tuberculosis. We aimed to assess the validity of sputum culture conversion on solid media at varying timepoints, and the time to conversion, as prognostic markers for end-of-treatment outcome in patients with multidrug-resistant (MDR) tuberculosis. We analysed data from two large cohort studies of patients with MDR tuberculosis. We defined sputum culture conversion as two or more consecutive negative cultures from sputum samples obtained at least 30 days apart. To estimate the association of 2 month and 6 month conversion with successful treatment outcome, we calculated odds ratios (ORs) and 95% CIs with random-effects multivariable logistic regression. We calculated predictive values with bivariate random-effects generalised linear mixed modelling. We assessed data for 1712 patients who had treatment success, treatment failure, or who died. Among patients with treatment success, median time to sputum culture conversion was significantly shorter than in those who had poor outcomes (2 months [IQR 1-3] vs 7 months [3 to ≥24]; log-rank p<0·0001). Furthermore, conversion status at 6 months (adjusted OR 14·07 [95% CI 10·05-19·71]) was significantly associated with treatment success compared with failure or death. Sputum culture conversion status at 2 months was significantly associated with treatment success only in patients who were HIV negative (adjusted OR 4·12 [95% CI 2·25-7·54]) or who had unknown HIV infection (3·59 [1·96-6·58]), but not in those who were HIV positive (0·38 [0·12-1·18]). Thus, the overall association of sputum culture conversion with a successful outcome was substantially greater at 6 months than at 2 months. 2 month conversion had low sensitivity (27·3% [95% confidence limit 16·6-41·4]) and high specificity (89·8% [82·3-94·4]) for prediction of treatment success. Conversely, 6 month sputum culture conversion status had high sensitivity (91·8% [85·9-95·4]), but moderate specificity (57·8% [42·5-71·6]). The maximum combined sensitivity and specificity for sputum culture conversion was reached between month 6 and month 10 of treatment. Time to sputum culture conversion, conversion status at 6 months, and conversion status at 2 months in patients without known HIV infection can be considered as proxy markers of end-of-treatment outcome in patients with MDR tuberculosis, although the overall association with treatment success is substantially stronger for 6 month than for 2 month conversion status. Investigators should consider these results regarding the validity of sputum culture conversion at various timepoints as an early predictor of treatment efficacy when designing phase 2 studies before investing substantial resources in large, long-term, phase 3 trials of new treatments for MDR tuberculosis. US Agency for International Development, US Centers for Disease Control and Prevention, Division of Intramural Research of the US National Institute of Allergy and Infectious Diseases, Korea Centers for Disease Control and Prevention. Copyright © 2015 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd.. All rights reserved.

Sputum Anticitrullinated Protein Antibodies in Patients With Long-standing Rheumatoid Arthritis.

PubMed

Polachek, Ari; Vree Egberts, Wilma; Fireman, Elizabeth; Druckman, Ido; Stark, Moshe; Paran, Daphna; Kaufman, Ilana; Wigler, Irena; Levartovsky, David; Caspi, Dan; Pruijn, Ger J M; Elkayam, Ori

2018-04-01

The aim of this study was to evaluate the presence of autoantibodies to cyclic citrullinated synthetic peptides (ACPAs) in the sputum of patients with long-standing rheumatoid arthritis (RA). Nineteen consecutive RA patients and 16 age- and sex-matched control subjects participated in this cross-sectional study. All underwent complete lung function tests and provided induced sputum. Antibodies to citrullinated (CitP) and the corresponding norleucine-containing (NorP) peptides in the sputum of the RA patients and control subjects, as well as in the serum of the RA patients, were determined by enzyme-linked immunosorbent assay. Patients with RA had the following characteristics: mean disease duration of 12 years, Disease Activity Score for 28 joints of 3.44, and Sharp-van der Heijde score of 57.5. Ten of the 19 RA patients showed high titers of ACPAs in their sera. Four of the seropositive (40%), none of the seronegative RA patients, and only 1 of the control subjects showed detectable levels of ACPAs in their sputum. The ratio between the reactivity with CitP and NorP peptides in the sputum was significantly higher in RA sputum than in control sputum (1.33 ± 1.2 vs. 0.64 ± 0.14, P = 0.02). A positive correlation was found between sputum ACPAs and age, serum ACPAs, sputum anti-NorP, serum anti-CitP/NorP reactivity ratio, and the proportion of neutrophils and lymphocytes in the sputum. No significant correlation was found between sputum ACPAs and disease severity, history of smoking, lung function tests, or treatment for RA. Anticitrullinated protein/peptide antibodies can be detected in the sputum of RA patients and are correlated with the presence in the serum.

A Prospective Evaluation of Circulating Tumor Cells and Cell-Free DNA in EGFR-Mutant Non-Small Cell Lung Cancer Patients Treated with Erlotinib on a Phase II Trial.

PubMed

Yanagita, Masahiko; Redig, Amanda J; Paweletz, Cloud P; Dahlberg, Suzanne E; O'Connell, Allison; Feeney, Nora; Taibi, Myriam; Boucher, David; Oxnard, Geoffrey R; Johnson, Bruce E; Costa, Daniel B; Jackman, David M; Jänne, Pasi A

2016-12-15

Genotype-directed therapy is the standard of care for advanced non-small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression. EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression. Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival. cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010-20. ©2016 AACR. ©2016 American Association for Cancer Research.

[Detection of KRAS mutation in colorectal cancer patients' cfDNA with droplet digital PCR].

PubMed

Luo, Yuwen; Li, Yao

2018-03-25

This study aims to develop a new method for the detection of KRAS mutations related to colorectal cancer in cfDNA, and to evaluate the sensitivity and accuracy of the detection. We designed a method of cfDNA based KRAS detection by droplets digital PCR (ddPCR). The theoretical performance of the method is evaluated by reference standard and compared to the ARMS PCR method. Two methods, ddPCR and qPCR, were successfully established to detect KRAS wild type and 7 mutants. Both methods were validated using plasmid standards and actual samples. The results were evaluated by false positive rate, linearity, and limit of detection. Finally, 52 plasma cfDNA samples from patients and 20 samples from healthy people were tested, the clinical sensitivity is 97.64%, clinical specificity is 81.43%. ddPCR method shows higher performance than qPCR. The LOD of ddPCR method reached single digits of cfDNA copies, it can detect as low as 0.01% to 0.04% mutation abundance.

Airway inflammation in iron ore miners exposed to dust and diesel exhaust.

PubMed

Adelroth, E; Hedlund, U; Blomberg, A; Helleday, R; Ledin, M-C; Levin, J O; Pourazar, J; Sandström, T; Järvholm, B

2006-04-01

The aim of the present study was to investigate if underground miners exposed to dust and diesel exhaust in an iron ore mine would show signs of airway inflammation as reflected in induced sputum. In total, 22 miners were studied, once after a holiday of at least 2 weeks and the second time after 3 months of regular work. Control subjects were 21 "white-collar" workers. All subjects completed a questionnaire regarding medical and occupational history, and underwent lung function testing and induced sputum collection. Total and differential cell counts and analyses of the fluid phase of the induced sputum were performed. Sampling of personal exposure to elemental carbon, nitrogen dioxide and inhalable dust was recorded. The average concentrations of inhalable dust, nitrogen dioxide and elemental carbon were 3.2 mg.m-3, 0.28 mg.m-3 and 27 microg.m-3, respectively. Miners had increased numbers of inflammatory cells, mainly alveolar macrophages and neutrophils, and increased concentrations of fibronectin, metalloproteinase-9 and interleukin-10 in induced sputum compared with controls. In conclusion, miners in an underground iron ore mine demonstrated persistent airway inflammation that was as pronounced after a 4-week holiday as after a 3-month period of work underground in the mine.

Semiquantitative Culture Analysis during Therapy for Mycobacterium avium Complex Lung Disease.

PubMed

Griffith, David E; Adjemian, Jennifer; Brown-Elliott, Barbara A; Philley, Julie V; Prevots, D Rebecca; Gaston, Christopher; Olivier, Kenneth N; Wallace, Richard J

2015-09-15

Microbiologically based criteria such as sputum culture conversion to negative have traditionally been used to define treatment success for mycobacterial diseases. There are, however, limited data regarding whether nontuberculous mycobacterial sputum culture conversion or semiquantitative culture analysis correlates with subjective or nonmicrobiologic objective indices of treatment response. To determine whether a semiquantitative mycobacterial culture scale correlated with clinical disease status and was predictive of long-term sputum mycobacterial culture conversion to negative in a cohort of patients with nodular/bronchiectatic Mycobacterium avium complex lung disease undergoing therapy. One hundred and eighty patients undergoing standard macrolide-based therapy for M. avium complex lung disease were monitored at standard frequent intervals with symptomatic, radiographic, and microbiologic data collected, including semiquantitative mycobacterial culture analysis. Analyses were used to evaluate clinical and microbiologic predictors of long-term sputum conversion to culture negative. After 12 months of therapy, 148 (82%) patients had sputum conversion to culture negative. Baseline semiquantitative sputum culture scores did not differ between patients with sputum conversion and those without. The change in sputum culture semiquantitative score from baseline to Month 3 was highly predictive of subsequent sputum long-term conversion status indicative of treatment success, as was improvement in cough, and especially early radiographic improvement. Early semiquantitative sputum agar plate culture results can be used to predict symptomatic and radiographic improvement as well as long-term sputum culture conversion to negative in this population. We suggest that semiquantitative sputum culture scores can be a useful tool for evaluating new nontuberculous mycobacterial lung disease therapies.

Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

PubMed Central

Anderson, Julia; Lemmer, Darrin; Lehmkuhl, Erik; Georghiou, Sophia B.; Heaton, Hannah; Wiggins, Kristin; Gillece, John D.; Schupp, James M.; Catanzaro, Donald G.; Crudu, Valeriu; Cohen, Ted; Rodwell, Timothy C.; Engelthaler, David M.

2016-01-01

Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool. PMID:27225403

Airway inflammation in Japanese COPD patients compared with smoking and nonsmoking controls

PubMed Central

Ishikawa, Nobuhisa; Hattori, Noboru; Kohno, Nobuoki; Kobayashi, Akihiro; Hayamizu, Tomoyuki; Johnson, Malcolm

2015-01-01

Purpose To assess the importance of inflammation in chronic obstructive pulmonary disease (COPD) by measuring airway and systemic inflammatory biomarkers in Japanese patients with the disease and relevant control groups. Patients and methods This was the first study of its type in Japanese COPD patients. It was a non-treatment study in which 100 participants were enrolled into one of three groups: nonsmoking controls, current or ex-smoking controls, and COPD patients. All participants underwent standard lung function assessments and provided sputum and blood samples from which the numbers of inflammatory cells and concentrations of biomarkers were measured, using standard procedures. Results The overall trends observed in levels of inflammatory cells and biomarkers in sputum and blood in COPD were consistent with previous reports in Western studies. Increasing levels of neutrophils, interleukin 8 (IL-8), surfactant protein D (SP-D), and Krebs von den Lungen 6 (KL-6) in sputum and clara cell 16 (CC-16), high-sensitivity C-reactive protein (hs-CRP), and KL-6 in serum and plasma fibrinogen were seen in the Japanese COPD patients compared with the non-COPD control participants. In sputum, significant correlations were seen between total cell count and matrix metalloproteinase 9 (MMP-9; P<0.001), neutrophils and MMP-9 (P<0.001), macrophages and KL-6 (P<0.01), total cell count and IL-8 (P<0.05), neutrophils and IL-8 (P<0.05), and macrophages and MMP-9 (P<0.05). Significant correlations were also observed between some inflammatory cells in sputum and biomarkers in serum, with the most significant between serum CC-16 and both total cell count (P<0.005) and neutrophils (P<0.005) in sputum. Conclusion These results provide evidence for the first time that COPD in Japanese patients is a multicomponent disease, involving both airway and systemic inflammation, in addition to airway obstruction. Therefore, intervention with anti-inflammatory therapy may provide additional benefit in disease management of COPD in Japan. PMID:25670894

Airway inflammation in Japanese COPD patients compared with smoking and nonsmoking controls.

PubMed

Ishikawa, Nobuhisa; Hattori, Noboru; Kohno, Nobuoki; Kobayashi, Akihiro; Hayamizu, Tomoyuki; Johnson, Malcolm

2015-01-01

To assess the importance of inflammation in chronic obstructive pulmonary disease (COPD) by measuring airway and systemic inflammatory biomarkers in Japanese patients with the disease and relevant control groups. This was the first study of its type in Japanese COPD patients. It was a non-treatment study in which 100 participants were enrolled into one of three groups: nonsmoking controls, current or ex-smoking controls, and COPD patients. All participants underwent standard lung function assessments and provided sputum and blood samples from which the numbers of inflammatory cells and concentrations of biomarkers were measured, using standard procedures. The overall trends observed in levels of inflammatory cells and biomarkers in sputum and blood in COPD were consistent with previous reports in Western studies. Increasing levels of neutrophils, interleukin 8 (IL-8), surfactant protein D (SP-D), and Krebs von den Lungen 6 (KL-6) in sputum and clara cell 16 (CC-16), high-sensitivity C-reactive protein (hs-CRP), and KL-6 in serum and plasma fibrinogen were seen in the Japanese COPD patients compared with the non-COPD control participants. In sputum, significant correlations were seen between total cell count and matrix metalloproteinase 9 (MMP-9; P<0.001), neutrophils and MMP-9 (P<0.001), macrophages and KL-6 (P<0.01), total cell count and IL-8 (P<0.05), neutrophils and IL-8 (P<0.05), and macrophages and MMP-9 (P<0.05). Significant correlations were also observed between some inflammatory cells in sputum and biomarkers in serum, with the most significant between serum CC-16 and both total cell count (P<0.005) and neutrophils (P<0.005) in sputum. These results provide evidence for the first time that COPD in Japanese patients is a multicomponent disease, involving both airway and systemic inflammation, in addition to airway obstruction. Therefore, intervention with anti-inflammatory therapy may provide additional benefit in disease management of COPD in Japan.

Potentially pathogenic airway bacteria and neutrophilic inflammation in treatment resistant severe asthma.

PubMed

Green, Benjamin J; Wiriyachaiporn, Surasa; Grainge, Christopher; Rogers, Geraint B; Kehagia, Valia; Lau, Laurie; Carroll, Mary P; Bruce, Kenneth D; Howarth, Peter H

2014-01-01

Molecular microbiological analysis of airway samples in asthma has demonstrated an altered microbiome in comparison to healthy controls. Such changes may have relevance to treatment-resistant severe asthma, particularly those with neutrophilic airway inflammation, as bacteria might be anticipated to activate the innate immune response, a process that is poorly steroid responsive. An understanding of the relationship between airway bacterial presence and dominance in severe asthma may help direct alternative treatment approaches. We aimed to use a culture independent analysis strategy to describe the presence, dominance and abundance of bacterial taxa in induced sputum from treatment resistant severe asthmatics and correlate findings with clinical characteristics and airway inflammatory markers. Induced sputum was obtained from 28 stable treatment-resistant severe asthmatics. The samples were divided for supernatant IL-8 measurement, cytospin preparation for differential cell count and Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling for bacterial community analysis. In 17/28 patients, the dominant species within the airway bacterial community was Moraxella catarrhalis or a member of the Haemophilus or Streptococcus genera. Colonisation with these species was associated with longer asthma disease duration (mean (SD) 31.8 years (16.7) vs 15.6 years (8.0), p = 0.008), worse post-bronchodilator percent predicted FEV1 (68.0% (24.0) vs 85.5% (19.7), p = 0.025) and higher sputum neutrophil differential cell counts (median (IQR) 80% (67-83) vs 43% (29-67), p = 0.001). Total abundance of these organisms significantly and positively correlated with sputum IL-8 concentration and neutrophil count. Airway colonisation with potentially pathogenic micro-organisms in asthma is associated with more severe airways obstruction and neutrophilic airway inflammation. This altered colonisation may have a role in the development of an asthma phenotype that responds less well to current asthma therapies.

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology▿ † ‡

PubMed Central

Helb, Danica; Jones, Martin; Story, Elizabeth; Boehme, Catharina; Wallace, Ellen; Ho, Ken; Kop, JoAnn; Owens, Michelle R.; Rodgers, Richard; Banada, Padmapriya; Safi, Hassan; Blakemore, Robert; Lan, N. T. Ngoc; Jones-López, Edward C.; Levi, Michael; Burday, Michele; Ayakaka, Irene; Mugerwa, Roy D.; McMillan, Bill; Winn-Deen, Emily; Christel, Lee; Dailey, Peter; Perkins, Mark D.; Persing, David H.; Alland, David

2010-01-01

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h. PMID:19864480

Inflammatory responses to the occupational inhalation of metal fume.

PubMed

Palmer, K T; McNeill Love, R M C; McNeill-Love, R; Poole, J R; Coggon, D; Frew, A J; Linaker, C H; Shute, J K

2006-02-01

Occupational exposure to metal fume promotes a reversible increase in the risk of pneumonia, but by mechanisms which are unclear. To investigate, the current authors measured various markers of host defence function in welders and nonwelders. Induced sputum and venous blood samples were collected from 27 welders with regular long-term exposure to ferrous metal fume and 31 unexposed matched controls. In sputum, the present authors measured cell counts, the soluble and cellular iron concentration, and levels of interleukin-8, tumour necrosis factor-alpha, myeloperoxidase, matrix metalloproteinase-9, immunoglobulin (Ig)A, alpha(2)-macroglobulin and unsaturated iron-binding capacity. Blood samples were assayed for evidence of neutrophil activation and pneumococcal IgG antibodies. Welders had significantly higher iron levels and a substantially lower unsaturated iron-binding capacity in their sputum, but, despite a high iron challenge, there was a noteworthy absence of an inflammatory response. Only blood counts of eosinophils and basophils were significantly related to the extent of welding. Weak nonsignificant trends were observed for several other measures, consistent with low-grade priming of neutrophils. In conclusion, these data suggest that chronic exposure to metal fume blunts responsiveness to inhaled particulate matter. However, the mechanism behind the lack of detectable local inflammatory response requires further investigation.

[Airway metaplasia related to smoking and environmental pollution through sputum].

PubMed

Ferman-Cano, Floribel; Padilla-Santamaría, Fernando; Moreno-Venegas, Luis; Torner-Aguilar, Carlos Alejandro; Molina-Medina, Miguel Angel

2018-01-01

Both environmental pollution and smoking affect the respiratory epithelium, causing cellular adaptation changes. Therefore, this work explores the presence of metaplasia in smokers compared with non-smokers from Mexico City. A prospective cohort study was performed. The sample was performed through a home interview covering the five Mexico City zones delimited to the study and monitoring of air quality. We searched metaplastic cells and inflammation in sputum cytology stained with Papanicolaou technique, and we assessed the air quality in Mexico City. We calculated relative risk (RR) and attributable risk (AR) in relation to the presence of metaplasia and smoking. We performed 1897 home interviews obtaining a final sample of 30 participants. There were no significant differences between smokers and non-smokers with the presence of metaplastic cells (p = 0.269), although the association of metaplastic cells and inflammation showed a significant difference in the non-smokers group (p = 0.010). The RR in association with the presence of metaplasia in sputum cytology and smoking was 1.6, and the RA was 0.2. The air quality in this city has led the population to undergo changes of cellular adaptation in the respiratory epithelium by the simple fact of being exposed to environmental pollution. Metaplastic changes in non-smokers suggest strongly that pollution causes the same effect as smoking.

Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

PubMed

Ho, Sherry Sze Yee; Barrett, Angela; Thadani, Henna; Asibal, Cecille Laureano; Koay, Evelyn Siew-Chuan; Choolani, Mahesh

2015-07-01

Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

[Value of optimization of bedside Gram staining of sputum smear in the early diagnosis and treatment of ventilator-associated pneumonia].

PubMed

Liao, Xinyan; Ran, Yu; Bian, Shichang; Wang, Chao; Xu, Lei

2014-12-01

To investigate the significance of optimization of bedside Gram staining of sputum smear in the early diagnosis and antimicrobial treatment for ventilator-associated pneumonia (VAP) patients. The data of patients with VAP undergoing mechanical ventilation over 48 hours in the Department of Critical Care Medicine of Tianjin Fourth Central Hospital from June 2009 to June 2014 were analyzed. The patients were divided into two groups according to whether or not bedside Gram staining of sputum smear was used or not. The sputum samples from lower respiratory tract of all VAP patients were collected daily with tracheal catheter. In empirical examination group (from June 2009 to December 2011, n=43), the patients received antibiotics at the time of onset of VAP, selection of antibiotics depended on the information of bacterial epidemiology of the intensive care unit (ICU), and also existence of high risk factors of multi-drug resistant bacteria. In target treatment group (from January 2012 to June 2014, n=43), the patients received antibiotics according to the results of bedside instant sputum smear examination and empirical antibiotic regime. The correlation between the results of sputum smear examination and culture result was analyzed. The levels of body temperature, white blood cell (WBC) count, procalcitonin (PCT) level,and high sensitivity C- reactive protein (hs-CRP) were measured on the 1st day and 3rd day. The length of antibiotics treatment, duration of mechanical ventilation, and the time of ICU stay were recorded for both groups. There were 512 qualified sputum specimens for culture, from which 336 pathogens were found, and 358 strains of pathogenic bacteria were found from microscopic examination of 512 qualified sputum smear. The coincidence rate of results of bedside examination of sputum smear and that of sputum culture was 78.32%(401/512). The diagnostic acumen of the former was 85.42% (287/336), specificity was 64.77% (114/176), positive predictive value was 80.17% (287/358), and negative predictive value was 74.03% (114/154). On the 1st day, no statistical differences in infection index between the two groups could be found, but on the 3rd day, the results were significantly improved in both groups. Compared with the empirical treatment group, the body temperature, WBC, PCT and hs-CRP in the target treatment group were significantly lower [body temperature (centigrade): 36.83 ± 0.69 vs. 37.64 ± 0.71, WBC (× 10⁹/L): 7.91 ± 2.75 vs. 9.66 ± 3.39, PCT (μg/L): 7.14 ± 3.89 vs. 10.14 ± 4.32, hs-CRP (mg/L): 12.24 ± 6.28 vs. 15.54 ± 5.94, P<0.05 or P<0.01]. Compared with the empirical treatment group, the time of antibiotics use(days: 6.00 ± 2.55 vs. 9.20 ± 3.46), the duration of mechanical ventilation (days: 5.00 ± 1.73 vs. 7.00 ± 1.94), and the length of ICU stay (days: 7.43 ± 1.72 vs. 12.57 ± 4.16) were significantly shortened (P<0.05 or P<0.01). The results of bedside sputum examination and sputum culture showed a good correlation, and the former is helpful in early diagnosis and treatment of VAP. The result of high quality sputum smear in significant in guiding the first choice of antibiotics, reduce the time of antibiotic use, shorten the duration of mechanical ventilation and the length of ICU stay, and improve the outcome of the patients.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khanal, Suraj P.; Mahfuz, Hassan; Rondinone, Adam Justin

The potential of improving the fracture toughness of synthetic hydroxyapatite (HAp) by incorporating carboxyl functionalized single walled carbon nanotubes (CfSWCNTs) and polymerized ε-caprolactam (nylon) was researched. A series of HAp samples with CfSWCNTs concentrations varying from 0 to 1.5 wt.%, without, and with nylon addition was prepared. X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) were used to characterize the samples. The three point bending test was applied to measure the fracture toughness of the composites. A reproducible value of 3.6 ± 0.3 MPa.√m was found for samples containing 1 wt.% CfSWCNTs and nylon. This valuemore » is in the range of the cortical bone fracture toughness. Lastly, the increase of the CfSWCNTs content results to decrease of the fracture toughness, and formation of secondary phases.« less

Multiplex real time PCR panels to identify fourteen colonization factors of enterotoxigenic Escherichia coli (ETEC).

PubMed

Liu, Jie; Silapong, Sasikorn; Jeanwattanalert, Pimmada; Lertsehtakarn, Paphavee; Bodhidatta, Ladaporn; Swierczewski, Brett; Mason, Carl; McVeigh, Annette L; Savarino, Stephen J; Nshama, Rosemary; Mduma, Esto; Maro, Athanasia; Zhang, Jixian; Gratz, Jean; Houpt, Eric R

2017-01-01

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.

Application of loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of pathogenic bacteria in clinical sputum specimens of acute exacerbation of COPD (AECOPD).

PubMed

Zhang, Wei; Chen, Chuanhui; Cui, Jian; Bai, Wei; Zhou, Jing

2015-01-01

The present study explores the application of LAMP for rapid diagnosis of pathogenic bacteria in clinical sputum specimens of AECOPD as compared with conventional sputum culturing method. 120 sputum specimens of AECOPD patients, 46 sputum specimens of healthy controls, as well as 166 serum specimens as negative controls, were evaluated by LAMP assay using primers of eight typical respiratory pathogens. No cross-reactivity was observed in these negative control species using LAMP assay. The lower detection limit of LAMP assay was approximately 10(3) copies. 25 cases (20.8%) were detected at least one positive bacteria species by conventional sputum culturing method, while 73 cases (60.8%) were tested positive in LAMP assay. Moreover, compared with sputum culture, bacterial titers results of LAMP assay were more consistent with FEV1/FVC value of AECOPD patients. These results indicated that the sensitivity of LAMP assay was significantly higher than that of sputum culturing method.

High rhinovirus burden in lower airways of children with cystic fibrosis.

PubMed

Kieninger, Elisabeth; Singer, Florian; Tapparel, Caroline; Alves, Marco P; Latzin, Philipp; Tan, Hui-Leng; Bossley, Cara; Casaulta, Carmen; Bush, Andrew; Davies, Jane C; Kaiser, Laurent; Regamey, Nicolas

2013-03-01

Rhinovirus (RV)-induced pulmonary exacerbations are common in cystic fibrosis (CF) and have been associated with impaired virus clearance by the CF airway epithelium in vitro. Here, we assess in vivo the association of RV prevalence and load with antiviral defense mechanisms, airway inflammation, and lung function parameters in children with CF compared with a control group and children with other chronic respiratory diseases. RV presence and load were measured by real-time reverse transcription-polymerase chain reaction in BAL samples and were related to antiviral and inflammatory mediators measured in BAL and to clinical parameters. BAL samples were obtained from children with CF (n = 195), non-CF bronchiectasis (n = 40), or asthma (n = 29) and from a control group (n = 35) at a median (interquartile range [IQR]) age of 8.2 (4.0-11.7) years. RV was detected in 73 samples (24.4%). RV prevalence was similar among groups. RV load (median [IQR] x 10(3) copies/mL) was higher in children with CF (143.0 [13.1-1530.0]), especially during pulmonary exacerbations, compared with children with asthma (3.0 [1.3-25.8], P = .006) and the control group (0.5 [0.3-0.5], P < .001), but similar to patients with non-CF bronchiectasis (122.1 [2.7-4423.5], P = not significant). In children with CF, RV load was negatively associated with interferon (IFN)- b and IFN- l , IL-1ra levels, and FEV 1 , and positively with levels of the cytokines CXCL8 and CXCL10. RV load in CF BAL is high, especially during exacerbated lung disease. Impaired production of antiviral mediators may lead to the high RV burden in the lower airways of children with CF. Whether high RV load is a cause or a consequence of inflammation needs further investigation in longitudinal studies.

Identification and validation of nebulized aerosol devices for sputum induction

PubMed Central

Davidson, Warren J; Dennis, John; The, Stephanie; Litoski, Belinda; Pieron, Cora; Leigh, Richard

2014-01-01

Induced sputum cell count measurement has proven reliability for evaluating airway inflammation in patients with asthma and other airway diseases. Although the use of nebulizer devices for sputum induction is commonplace, they are generally labelled as single-patient devices by the manufacturer and, therefore, cannot be used for multiple patients in large clinical sputum induction programs due to infect ion-control requirements. Accordingly, this study investigated the aerosol characteristics of alternative devices that could be used in such programs. BACKGROUND: Induced sputum cell counts are a noninvasive and reliable method for evaluating the presence, type and degree of airway inflammation in patients with asthma. Currently, standard nebulizer devices used for sputum induction in multiple patients are labelled as single-patient devices by the manufacturer, which conflicts with infection prevention and control requirements. As such, these devices cannot feasibly be used in a clinical sputum induction program. Therefore, there is a need to identify alternative nebulizer devices that are either disposable or labelled for multi-patient use. OBJECTIVE: To apply validated rigorous, scientific testing methods to identify and validate commercially available nebulizer devices appropriate for use in a clinical sputum induction program. METHODS: Measurement of nebulized aerosol output and size for the selected nebulizer designs followed robust International Organization for Standardization methods. Sputum induction using two of these nebulizers was successfully performed on 10 healthy adult subjects. The cytotechnologist performing sputum cell counts was blinded to the type of nebulizer used. RESULTS: The studied nebulizers had variable aerosol outputs. The AeroNeb Solo (Aerogen, Ireland), Omron NE-U17 (Omron, Japan) and EASYneb II (Flaem Nuova, Italy) systems were found to have similar measurements of aerosol size. There was no significant difference in induced sputum cell results between the AeroNeb Solo and EASYneb II devices. DISCUSSION: There is a need for rigorous, scientific evaluation of nebulizer devices for clinical applications, including sputum induction, for measurement of cell counts. CONCLUSION: The present study was the most comprehensive analysis of different nebulizer devices for sputum induction to measure cell counts, and provides a framework for appropriate evaluation of nebulizer devices for induced sputum testing. PMID:24288700

Colour segmentation of multi variants tuberculosis sputum images using self organizing map

NASA Astrophysics Data System (ADS)

Rulaningtyas, Riries; Suksmono, Andriyan B.; Mengko, Tati L. R.; Saptawati, Putri

2017-05-01

Lung tuberculosis detection is still identified from Ziehl-Neelsen sputum smear images in low and middle countries. The clinicians decide the grade of this disease by counting manually the amount of tuberculosis bacilli. It is very tedious for clinicians with a lot number of patient and without standardization for sputum staining. The tuberculosis sputum images have multi variant characterizations in colour, because of no standardization in staining. The sputum has more variants colour and they are difficult to be identified. For helping the clinicians, this research examined the Self Organizing Map method for colouring image segmentation in sputum images based on colour clustering. This method has better performance than k-means clustering which also tried in this research. The Self Organizing Map could segment the sputum images with y good result and cluster the colours adaptively.

Electrophoretically-Deposited Nano-Fe3O4@carbon 3D Structure on Carbon Fiber as High-Performance Supercapacitors

NASA Astrophysics Data System (ADS)

Hajalilou, Abdollah; Etemadifar, Reza; Abbasi-Chianeh, Vahid; Abouzari-Lotf, Ebrahim

2018-05-01

Structural and electrochemical behaviors of electrophortically-deposited Fe3O4 and Fe3O4@C nanoparticles on carbon fiber (CF) were investigated. The nanoparticles were synthesized via a green-assisted hydrothermal route. The as-prepared samples were characterized by x-ray diffraction, transmission and scanning electron microscopies, Fourier transform infrared and UV-visible spectroscopies as well as by a vibration sample magnetometer. Surprisingly, the saturation magnetization (M s) of the Fe3O4@C ( 26.99 emu/g) was about 20% higher than that of Fe3O4 nanoparticles. A rather rectangular CV curve for both the elecrophortically-deposited Fe3O4 and Fe3O4@C on CF indicated the double-layer supercapacitor behavior of the samples. The synergistic effects of double shells improved the electrochemical behavior of Fe3O4@CF. The Fe3O4@C@CF composite exhibited a higher specific capacitance of 412 F g-1 at scan rate of 0.05 V/s compared to the Fe3O4@CF with a value of 193 F g-1. The superb electrochemical properties of Fe3O4@C@CF confirm their potential for applications as supercapacitors in the energy storage field.

Microbiological quality of complementary foods and its association with diarrhoeal morbidity and nutritional status of Bangladeshi children.

PubMed

Islam, M A; Ahmed, T; Faruque, A S G; Rahman, S; Das, S K; Ahmed, D; Fattori, V; Clarke, R; Endtz, H P; Cravioto, A

2012-11-01

To examine the bacteriological quality of complementary foods (CF) and to correlate the results with diarrhoeal morbidity and nutritional status of Bangladeshi children aged 6-24 months. A total of 212 CF samples were tested, of which 72 were collected immediately before the first time of feeding (≤ 1 h of food preparation) and 140 were collected at second/third time of feeding from 140 households located in urban and rural areas of Bangladesh. Anthropometry, food frequency data and demographic information of the children were collected. Of the first time feeding samples, 3% from each of urban and rural areas were found to be contaminated with faecal coliforms (FC) at ≥ 100 CFU/g. E. coli was isolated from 11% and 6% of samples, and B. cereus from 8% and 6% of samples from urban and rural areas, respectively. In contrast, 33% of the second/third time feeding samples from urban areas and 19% from rural areas were contaminated with FC at ≥ 100 CFU/g (P<0.05). E. coli was isolated from 40% and 39% of samples, and B. cereus from 33% and 26% of samples from urban and rural areas, respectively. Significantly high numbers of wasted rural children had CF with a high aerobic plate count, which was also significantly associated with diarrhoeal morbidity in children. Around 40% of CF samples were contaminated with E. coli, which was mainly attributable to food preparation practices. Consumption of contaminated CF appeared to be associated with a higher frequency of diarrhoea and malnutrition in children.

Familial concordance of phenotype and microbial variation among siblings with CF.

PubMed

Picard, Elie; Aviram, Micha; Yahav, Yaakov; Rivlin, Joseph; Blau, Hanna; Bentur, Lea; Avital, Avraham; Villa, Yael; Schwartz, Shepard; Kerem, Batsheva; Kerem, Eitan

2004-10-01

The clinical spectrum of cystic fibrosis (CF) is influenced by the cystic fibrosis transmembrane conductance regulator (CFTR) genotype. However, variable courses of the disease were demonstrated among patients with identical genotypes. Since siblings share identical CFTR mutations and environmental factors, they can serve as a model to assess the effect of modifier genes on disease expression, and also to evaluate cross-infection. The aim of this study was to compare disease expression among siblings with CF. All sibling pairs treated at 7 CF centers in Israel were included in the study. Data were collected from patients' medical charts. Fifty families with at least 2 siblings were identified. As expected, the second-born sibling was diagnosed at an earlier age compared to the first-born. The mode of CF presentation at diagnosis showed significant familial concordance. In the families where the first sibling presented with gastrointestinal manifestations, 79% of the second siblings also presented with gastrointestinal manifestations. When gastrointestinal manifestations were absent in the first sibling, only 12% of the second siblings presented with gastrointestinal manifestations (P < 0.0001). Likewise, when the first sibling presented with respiratory symptoms, 60% of the second siblings presented with the similar symptoms. However, when the first sibling presented without respiratory symptoms, only 12% of the second siblings presented with respiratory symptoms (P < 0.001). Meconium ileus (MI) was present in 20 patients (21%). In 10 families where the first-born sibling had MI, 8 (80%) of the subsequent siblings had MI. On the other hand, in the 39 families where the first-born sibling did not have MI, only 2 (5%) subsequent siblings had MI (P < 0.001). Pancreatic insufficiency (PI) also had high familial concordance (P < 0.0001). Percentile growth for weights and heights and lung function (FVC, FEV(1), and FEF(25-75)) at ages 7 and 10 years were similar between siblings. P. aeruginosa grew from sputum in 89% of our study patients. When P. aeruginosa was isolated from the first-born patient, 91% of the second siblings were also positive for P. aeruginosa, whereas when the initial sibling was not a carrier of P. aeruginosa, only 50% of subsequent siblings were positive (P < 0.0001). This familial concordance was not observed for S. aureus. By contrast, the age of first isolation of P. aeruginosa and S. aureus was significantly earlier in the second sibling than in the first for the two bacteria: 10.3 +/- 5.1 vs. 7.3 +/- 5.2 years (P < 0.05) for P. aeruginosa, and 11.5 +/- 5.4 years vs. 6.8 +/- 5.1 years (P < 0.05) for S. aureus. CF siblings tend to share similar phenotypes that are not mutation-dependent. The lack of variability between siblings in mode of initial CF presentation, rates of MI, pulmonary function, and nutritional status supports the role of modifier genes in the determination of these factors.

Carotid-femoral pulse wave velocity in a healthy adult sample: The ELSA-Brasil study.

PubMed

Baldo, Marcelo Perim; Cunha, Roberto S; Molina, Maria Del Carmen B; Chór, Dora; Griep, Rosane H; Duncan, Bruce B; Schmidt, Maria Inês; Ribeiro, Antonio L P; Barreto, Sandhi M; Lotufo, Paulo A; Bensenor, Isabela M; Pereira, Alexandre C; Mill, José Geraldo

2018-01-15

Aging declines essential physiological functions, and the vascular system is strongly affected by artery stiffening. We intended to define the age- and sex-specific reference values for carotid-to-femoral pulse wave velocity (cf-PWV) in a sample free of major risk factors. The ELSA-Brasil study enrolled 15,105 participants aged 35-74years. The healthy sample was achieved by excluding diabetics, those over the optimal and normal blood pressure levels, body mass index ≤18.5 or ≥25kg/m 2 , current and former smokers, and those with self-report of previous cardiovascular disease. After exclusions, the sample consisted of 2158 healthy adults (1412 women). Although cf-PWV predictors were similar between sex (age, mean arterial pressure (MAP) and heart rate), cf-PWV was higher in men (8.74±1.15 vs. 8.31±1.13m/s; adjusted for age and MAP, P<0.001) for all age intervals. When divided by MAP categories, cf-PWV was significantly higher in those which MAP ≥85mmHg, regardless of sex, and for all age intervals. Risk factors for arterial stiffening in the entire ELSA-Brasil population (n=15,105) increased by twice the age-related slope of cf-PWV growth, regardless of sex (0.0919±0.182 vs. 0.0504±0.153m/s per year for men, 0.0960±0.173 vs. 0.0606±0.139m/s per year for women). cf-PWV is different between men and women and even in an optimal and normal range of MAP and free of other classical risk factors for arterial stiffness, reference values for cf-PWV should take into account MAP levels. Also, the presence of major risk factors in the general population doubles the age-related rise in cf-PWV. Copyright © 2017 Elsevier B.V. All rights reserved.

Pharmacodynamic Modeling of Bacillary Elimination Rates and Detection of Bacterial Lipid Bodies in Sputum to Predict and Understand Outcomes in Treatment of Pulmonary Tuberculosis

PubMed Central

Sloan, Derek J.; Mwandumba, Henry C.; Garton, Natalie J.; Khoo, Saye H.; Butterworth, Anthony E.; Allain, Theresa J.; Heyderman, Robert S.; Corbett, Elizabeth L.; Barer, Mike R.; Davies, Geraint R.

2015-01-01

Background. Antibiotic-tolerant bacterial persistence prevents treatment shortening in drug-susceptible tuberculosis, and accumulation of intracellular lipid bodies has been proposed to identify a persister phenotype of Mycobacterium tuberculosis cells. In Malawi, we modeled bacillary elimination rates (BERs) from sputum cultures and calculated the percentage of lipid body–positive acid-fast bacilli (%LB + AFB) on sputum smears. We assessed whether these putative measurements of persistence predict unfavorable outcomes (treatment failure/relapse). Methods. Adults with pulmonary tuberculosis received standard 6-month therapy. Sputum samples were collected during the first 8 weeks for serial sputum colony counting (SSCC) on agar and time-to positivity (TTP) measurement in mycobacterial growth indicator tubes. BERs were extracted from nonlinear and linear mixed-effects models, respectively, fitted to these datasets. The %LB + AFB counts were assessed by fluorescence microscopy. Patients were followed until 1 year posttreatment. Individual BERs and %LB + AFB counts were related to final outcomes. Results. One hundred and thirty-three patients (56% HIV coinfected) participated, and 15 unfavorable outcomes were reported. These were inversely associated with faster sterilization phase bacillary elimination from the SSCC model (odds ratio [OR], 0.39; 95% confidence interval [CI], .22–.70) and a faster BER from the TTP model (OR, 0.71; 95% CI, .55–.94). Higher %LB + AFB counts on day 21–28 were recorded in patients who suffered unfavorable final outcomes compared with those who achieved stable cure (P = .008). Conclusions. Modeling BERs predicts final outcome, and high %LB + AFB counts 3–4 weeks into therapy may identify a persister bacterial phenotype. These methods deserve further evaluation as surrogate endpoints for clinical trials. PMID:25778753

The Cell-CT 3D Cell Imaging Technology Platform Enables the Detection of Lung Cancer Using the Non-Invasive LuCED Sputum Test

PubMed Central

Meyer, Michael G.; Hayenga, Jon; Neumann, Thomas; Katdare, Rahul; Presley, Chris; Steinhauer, David; Bell, Timothy; Lancaster, Christy; Nelson, Alan C.

2015-01-01

The war against cancer has yielded important advances in the early diagnosis and treatment of certain cancer types, but the poor detection rate and 5-year survival rate for lung cancer remains little changed over the past 40 years. Early detection through emerging lung cancer screening programs promises the most reliable means of improving mortality. Sputum cytology has been tried without success because sputum contains few malignant cells that are difficult for cytologists to detect. However, research has shown that sputum contains diagnostic malignant cells and could serve as a means of lung cancer detection if those cells could be detected and correctly characterized. Recently, the National Lung Cancer Screening Trial reported that screening by three consecutive low-dose X-ray CT scans provides a 20% reduction in lung cancer mortality compared to chest X-ray. This reduction in mortality, however, comes with an unacceptable false positive rate that increases patient risks and the overall cost of lung cancer screening. This article reviews the LuCED® test for detecting early lung cancer. LuCED is based on patient sputum that is enriched for bronchial epithelial cells. The enriched sample is then processed on the Cell-CT®, which images cells in three dimensions with sub-micron resolution. Algorithms are applied to the 3D cell images to extract morphometric features that drive a classifier to identify cells that have abnormal characteristics. The final status of these candidate abnormal cells is established by the pathologist's manual review. LuCED promotes accurate cell classification which could enable cost effective detection of lung cancer. PMID:26148817

Responsiveness of blood and sputum inflammatory cells in Japanese COPD patients, non-COPD smoking controls, and non-COPD nonsmoking controls

PubMed Central

Kawayama, Tomotaka; Kinoshita, Takashi; Matsunaga, Kazuko; Kobayashi, Akihiro; Hayamizu, Tomoyuki; Johnson, Malcolm; Hoshino, Tomoaki

2016-01-01

Purpose To compare pulmonary and systemic inflammatory mediator release, pre- and poststimulation, ex vivo, in cells from Japanese patients with chronic obstructive pulmonary disease (COPD), non-COPD smoking controls, and non-COPD nonsmoking controls (NSC). Patients and methods This was a nontreatment study with ten subjects per group. Inflammatory biomarker release, including interleukin (IL)-6 and -8, matrix metalloproteinase-9, and tumor necrosis factor (TNF)-α, was measured in peripheral blood mononuclear cells (PBMC) and sputum cells with and without lipopolysaccharide or TNF-α stimulation. Results In PBMC, basal TNF-α release (mean ± standard deviation) was significantly different between COPD (81.6±111.4 pg/mL) and nonsmoking controls (9.5±5.2 pg/mL) (P<0.05). No other significant differences were observed. Poststimulation biomarker release tended to increase, with the greatest changes in the COPD group. The greatest mean increases were seen in the lipopolysaccharide-induced release of matrix metalloproteinase-9, TNF-α, and IL-6 from PBMC. Pre- and poststimulation data from sputum samples were more variable and less conclusive than from PBMC. In the COPD group, induced sputum neutrophil levels were higher and macrophage levels were lower than in either control group. Significant correlations were seen between the number of sputum cells (macrophages and neutrophils) and biomarker levels (IL-8, IL-6, and TNF-α). Conclusion This was the first study to compare cellular inflammatory mediator release before and after stimulation among Japanese COPD, smoking controls, and nonsmoking controls populations. Poststimulation levels tended to be higher in patients with COPD. The results suggest that PBMC are already preactivated in the circulation in COPD patients. This provides further evidence that COPD is a multicomponent disease, involving both airway and systemic inflammation. PMID:26929615

Responsiveness of blood and sputum inflammatory cells in Japanese COPD patients, non-COPD smoking controls, and non-COPD nonsmoking controls.

PubMed

Kawayama, Tomotaka; Kinoshita, Takashi; Matsunaga, Kazuko; Kobayashi, Akihiro; Hayamizu, Tomoyuki; Johnson, Malcolm; Hoshino, Tomoaki

2016-01-01

To compare pulmonary and systemic inflammatory mediator release, pre- and poststimulation, ex vivo, in cells from Japanese patients with chronic obstructive pulmonary disease (COPD), non-COPD smoking controls, and non-COPD nonsmoking controls (NSC). This was a nontreatment study with ten subjects per group. Inflammatory biomarker release, including interleukin (IL)-6 and -8, matrix metalloproteinase-9, and tumor necrosis factor (TNF)-α, was measured in peripheral blood mononuclear cells (PBMC) and sputum cells with and without lipopolysaccharide or TNF-α stimulation. In PBMC, basal TNF-α release (mean ± standard deviation) was significantly different between COPD (81.6±111.4 pg/mL) and nonsmoking controls (9.5±5.2 pg/mL) (P<0.05). No other significant differences were observed. Poststimulation biomarker release tended to increase, with the greatest changes in the COPD group. The greatest mean increases were seen in the lipopolysaccharide-induced release of matrix metalloproteinase-9, TNF-α, and IL-6 from PBMC. Pre- and poststimulation data from sputum samples were more variable and less conclusive than from PBMC. In the COPD group, induced sputum neutrophil levels were higher and macrophage levels were lower than in either control group. Significant correlations were seen between the number of sputum cells (macrophages and neutrophils) and biomarker levels (IL-8, IL-6, and TNF-α). This was the first study to compare cellular inflammatory mediator release before and after stimulation among Japanese COPD, smoking controls, and nonsmoking controls populations. Poststimulation levels tended to be higher in patients with COPD. The results suggest that PBMC are already preactivated in the circulation in COPD patients. This provides further evidence that COPD is a multicomponent disease, involving both airway and systemic inflammation.

The Arginine Decarboxylase Pathways of Host and Pathogen Interact to Impact Inflammatory Pathways in the Lung

PubMed Central

Dalluge, Joseph J.; Welchlin, Cole W.; Hughes, John; Han, Wei; Blackwell, Timothy S.; Laguna, Theresa A.; Williams, Bryan J.

2014-01-01

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic. PMID:25350753

The arginine decarboxylase pathways of host and pathogen interact to impact inflammatory pathways in the lung.

PubMed

Paulson, Nick B; Gilbertsen, Adam J; Dalluge, Joseph J; Welchlin, Cole W; Hughes, John; Han, Wei; Blackwell, Timothy S; Laguna, Theresa A; Williams, Bryan J

2014-01-01

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.

Association between protozoa in sputum and asthma: a case-control study.

PubMed

van Woerden, Hugo C; Ratier-Cruz, Adriana; Aleshinloye, Olabode B; Martinez-Giron, Rafael; Gregory, Clive; Matthews, Ian P

2011-06-01

Atypical infectious agents have been proposed as potential contributors to asthma. A novel set of morphological and staining criteria permit the identification of flagellated protozoa in sputum. This case-control study was designed to use this novel method and to assess: (1) are protozoa more common in asthmatics than in non-asthmatics; (2) is the presence of protozoa associated with the use of steroid inhalers; and (3) is the presence of protozoa associated with living in damp housing? Induced sputum samples were collected from asthma patients and local non-atopic, non-smoking controls. Questionnaires assessed asthma severity and housing conditions. Sputum was examined for flagellated protozoa using a previously described staining technique. 96 participants were recruited for this study; 54 asthma patients and 42 controls, age range 21-62 years, 70% female participants. Limiting results to those who were clearly positive or negative for flagellated protozoa, 66.7% (20/30) of asthmatics and 30.8% (4/13) of controls had protozoa (p = 0.046). Among the asthma patients, prevalence of protozoa was not significantly different between those who had (10/18), and those who had not (10/12), used steroid inhaler in the preceding two weeks (p = 0.11). Similarly, the prevalence of protozoa was not significantly different between those who did (6/11) and those who did not (18/32), live in damp homes (p = 0.92). This case-control study demonstrates an association between flagellated protozoa in sputum and asthma. It is now necessary to confirm and characterise the protozoa using genetic techniques based on 18S ribosomal RNA. Once tis is established it would be worthwhile to determine if asthma symptoms improve when treated by anti-protozoal agents. Copyright © 2010 Elsevier Ltd. All rights reserved.

CF6-6D engine short-term performance deterioration

NASA Technical Reports Server (NTRS)

Kramer, W. H.; Paas, J. E.; Smith, J. J.; Wulf, R. H.

1980-01-01

Studies conducted as part of the NASA-Lewis CF6 jet engine diagnostics program are summarized. An 82-engine sample of DC-10-10 aircraft engine checkout data that were gathered to define the extent and magnitude of CF6-6D short term performance deterioration were analyzed. These data are substantiated by the performance testing and analytical teardown of CF6-6D short term deterioration engine serial number (ESN) 451507.

Smear-positive pulmonary tuberculosis and AFB examination practices according to the standard checklist of WHO's tuberculosis laboratory assessment tool in three governmental hospitals, Eastern Ethiopia.

PubMed

Mekonnen, Abiyu

2014-05-13

Using the Directly Observed Treatment-Short course (DOTS) program the World Health Organization's global target was to detect 70% of new sputum-smear positive PTB cases. Smear positive PTB cases are more infectious than the smear negative cases. The TB case detection rate remains very low in Ethiopia, but there are increases in smear-negative PTB diagnosis which could be attributed to several factors including poor quality of sputum smear-microscopy. A five years retrospective record review of data between September, 2007 and August, 2012 and an in-depth assessment of AFB staining practices of sputum smear using a standard checklist were made. The proportion of smear positive cases relative to overall Acid Fast Bacilli (AFB) screened was determined over a five year period to indicate the overall prevalence and the trend. Odds ratio with 95 percent confidence interval was calculated for categorical variables using multivariate Logistic Regression model to assess the strength of association. A total of 1266 individuals' data were reviewed. The majority of the study participants were male, 704 (55.6%), and rural residents, 690 (54.5%). The overall prevalence rate of smear positive PTB was 21.6%. Age categories between 15-24 and 25-34 years were independent predictors of smear positive PTB with adjusted odds ratio of 2.246 [95% CI (1.098-4.597)] and 2.267 [95% CI (1.107-4.642)], respectively. More males were affected by PTB than females with an adjusted odds ratio of 1.426 [95% CI (1.083-1.879)]. An in-depth interview with the respective laboratory chiefs showed that quality control measures for sputum smear microscopy were used at different levels of the testing activities; however, equipment function verification as a quality control measure was not accomplished regularly in all of the study hospital laboratories. The smear positive PTB case detection rate indicated in this study is significantly lower than the countries which met the 70% target of the World Health Organization. Lack of feedback mechanisms in the External Quality Assurance schemes of sputum smear microscopy render the opportunity for improvement difficult; Serial sputum examination showed a considerable rate of positivity in the second sputum sample when compared with the others.

Amniotic fluid digestive enzyme analysis is useful for identifying CFTR gene mutations of unclear significance.

PubMed

Oca, Florine; Dreux, Sophie; Gérard, Bénédicte; Simon-Bouy, Brigitte; de Becdelièvre, Alix; Ferec, Claude; Girodon, Emmanuelle; Muller, Françoise

2009-12-01

The large number of CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations and the existence of variants of unclear significance complicate the prenatal diagnosis of cystic fibrosis (CF). The aim of this study was to determine whether the pattern of amniotic fluid digestive enzymes (AF-DEs) could be correlated with the severity of CFTR mutations. The AF-DE pattern (gamma-glutamyltranspeptidase, aminopeptidase M, and the intestinal isoform of alkaline phosphatase) was retrospectively analyzed in 43 AF samples. All fetuses presented 2 CFTR mutations, which were classified according to the severity of the disease: CF/CF (n = 38); CF/CFTR-related disorders (n = 1); and CF/unknown variant (n = 4). The relationships between clinical CF status, CFTR mutations, and AF-DE pattern were studied. Of 38 severely affected CF fetuses, an "obstructive" AF-DE pattern was observed in 15 of 15 samples collected before 22 weeks, irrespective of the CFTR mutation (diagnostic sensitivity, 100%; diagnostic specificity, 99.8%). In the 23 fetuses evaluated after 22 weeks, the AF-DE pattern was abnormal in 7 cases and noncontributive in 16 (diagnostic sensitivity, 30.4%; diagnostic specificity, 99.8%). Of the 5 questionable cases (F508del/N1224K, F508del/L73F, 3849+10kbC>T/G1127E, F508del/S1235R, F508del/G622D), all were CF symptom free at 2-4 years of follow-up. The AF-DE pattern (<22 weeks) was typical in 3 cases but abnormal in the last 2 cases. AF-DE analysis is of value for prenatal CF diagnosis in classic forms of CF and could be helpful in nonclassic CF.

Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia.

PubMed

Letendre, Jo-Annie; Goggs, Robert

2017-05-01

To determine if cell-free DNA (cfDNA) was identifiable in canine plasma, to evaluate 3 techniques for the measurement of plasma cfDNA concentrations in dogs presented to an emergency service, and to compare the plasma cfDNA concentrations of healthy dogs to those with sepsis, trauma, and neoplasia. Retrospective study of banked canine plasma samples collected between May 2014 and December 2014. Dogs presented to the emergency service of a university veterinary teaching hospital. Plasma cfDNA was measured on residual plasma samples obtained from 15 dogs with sepsis, 15 dogs with moderate-severe trauma, 15 dogs diagnosed with a sarcoma. Plasma cfDNA was also measured in 15 healthy dogs. None. Assay linearity, repeatability, and reproducibility were evaluated. Quantification of cfDNA was performed in duplicate on diluted citrated plasma and following DNA purification using 2 fluorescence assays (SYBR-Gold; Quant-iT) and by ultraviolet absorbance spectroscopy. Fluorescence intensities (FIs) were converted to cfDNA concentrations using standard curves. Median FI values and cfDNA concentrations were compared to healthy controls using the Kruskal-Wallis test, with adjustment for multiple comparisons. Alpha was set at 0.05. Both assays had excellent linearity, and acceptable repeatability and reproducibility. Compared to controls, plasma cfDNA concentrations were significantly increased in dogs with sepsis or moderate-severe trauma with both assays (P ≤ 0.003). Dogs with neoplasia had significantly increased cfDNA concentrations with the Quant-iT assay only (P = 0.003). When measurements were performed on purified DNA, only dogs with moderate-severe trauma had significantly increased cfDNA concentrations (P < 0.001; SYBR-Gold assay). cfDNA can be readily identified in canine plasma using 2 fluorescence assays. DNA extraction offers no advantage over direct measurement. Compared to healthy controls, dogs with sepsis or moderate-severe trauma have significantly increased plasma cfDNA concentrations. © Veterinary Emergency and Critical Care Society 2017.

In vitro expression of Streptococcus pneumoniae ply gene in human monocytes and pneumocytes.

PubMed

Hu, Da-Kang; Liu, Yang; Li, Xiang-Yang; Qu, Ying

2015-05-07

Streptococcus pneumoniae is one major cause of pneumonia in human and contains various virulence factors that contribute to pathogenesis of pneumococcal disease. This study investigated the role of pneumolysin, Ply, in facilitating S. pneumoniae invasion into the host blood stream. S. pneumoniae strains were isolated from clinical blood and sputum samples and confirmed by PCR. Expression of ply gene was assessed by infecting human monocytes and pneumocytes. A total of 23 strains of S. pneumoniae isolated from blood (n = 11) and sputum (n = 12) along with S. pneumoniae ATCC49619 were used to infect human monocyte (THP-1) and Type II pneumocyte (A549) cell lines. All clinical strains of S. pneumoniae showed higher expression of ply mRNA than the American Type Culture Collection (ATCC) strain. Among the clinical strains, blood isolates showed higher expression of ply genes than sputum isolates, i.e., 2(1.5)- to 2(1.6)-folds in THP-1 and 2(0.4)- to 2(4.9)-folds in A549 cell lines. The data from the current study demonstrated that ply gene of blood- and sputum-derived S. pneumoniae is differentially expressed in two different cell lines. Under survival pressure, ply is highly expressed in these two cell lines for blood-derived S. pneumoniae, indicating that ply gene may facilitate S. pneumoniae invasion into the host blood system.

Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP.

PubMed

Intorasoot, S; Tharinjaroen, C S; Phunpae, P; Butr-Indr, B; Anukool, U; Intachai, K; Orrapin, S; Apiratmateekul, N; Arunothong, S; Suthachai, V; Saengsawang, K; Khamnoi, P; Pata, S; Kasinrerk, W; Tragoolpua, K

2016-08-01

To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens. Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively. The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens. This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden. © 2016 The Society for Applied Microbiology.

Role of 2-month sputum smears in predicting culture conversion in pulmonary tuberculosis.

PubMed

Su, W-J; Feng, J-Y; Chiu, Y-C; Huang, S-F; Lee, Y-C

2011-02-01

Sputum smears and culture conversion are frequently used to evaluate treatment response in pulmonary tuberculosis patients. Limited data are available on the evaluation of the correlation between under-treatment sputum smear results and culture conversion. This prospective study included sputum culture-proven pulmonary tuberculosis patients at six hospitals in Taiwan. At least two sets of sputum were collected at the completion of 8 weeks of TB treatment. The sensitivities and specificities of 2-month sputum smears were estimated based on culture conversion status. A total of 371 patients were enrolled for analysis. Factors associated with culture conversion included having a smear positive before treatment, presence of a cavity on radiography, rifampicin resistance and usage of the DOTS (directly observed therapy, short course) strategy. The sensitivities of 2-month sputum smears for culture conversion among all patients, initially smear-positive patients and initially smear-negative patients were 64.3, 71.4 and 38%, respectively, and the specificities were 81.6, 69.9 and 92.8%, respectively. In patients who were 2-month sputum smear-positive, the 2-month culture conversion rate was 80% if the patients were under DOTS and without cavitary lesions in radiograms. The predictive value of 2-month sputum smears in culture conversion was limited and highly influenced by clinical factors in pulmonary tuberculosis patients.

Evaluation of a rapid diagnostic test for the detection of Burkholderia pseudomallei in the Lao People's Democratic Republic.

PubMed

Woods, Kate L; Boutthasavong, Latsaniphone; NicFhogartaigh, Caoimhe; Lee, Sue J; Davong, Viengmon; AuCoin, David P; Dance, David A B

2018-05-02

Burkholderia pseudomallei causes significant global morbidity and mortality, with the highest disease burden in parts of Asia where culture-based diagnosis is often not available. We prospectively evaluated the Active Melioidosis Detect (AMD, InBios International, USA) lateral flow immunoassay (LFI) for rapid detection of B. pseudomallei in turbid blood cultures, pus, sputum, sterile fluid, urine, and sera. Performance was compared to B. pseudomallei detection using monoclonal antibody latex agglutination (LA) and immunofluorescence assays (IFA), with culture as the gold standard. AMD was 99% (99/100; 94.6 - 100%) sensitive and 100% (308/308; 98.8-100%) specific on turbid blood culture bottles, with no difference to LA or IFA. AMD specificity was 100% on pus (122/122; 97.0-100%), sputum (20/20; 83.2-100%), and sterile fluid (44/44; 92 - 100%). Sensitivity on these samples was: pus 47.1% (8/17; 23.0 - 72.2%), sputum 33.3% (1/3; 0.84 - 90.6%), and sterile fluid 0% (0/2; 0 - 84.2%). Urine AMD had a positive predictive value of 94% (32/34; 79.7 - 98.5%) for diagnosing melioidosis in our cohort. AMD sensitivity on stored sera, collected prospectively from melioidosis cases during this study, was 13.9% (5/36; 4.7% - 29.5%) when compared to blood culture samples taken on the same day. In conclusion, the AMD is an excellent tool for rapid diagnosis of melioidosis from turbid blood cultures, and maintains specificity across all sample types. It is a promising tool for urinary antigen detection, which could revolutionise diagnosis of melioidosis in resource-limited settings. Further work is required to improve sensitivity on non-blood culture samples. Copyright © 2018 Woods et al.

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification

PubMed Central

Peng, Peichao; Cheng, Xiaoxing; Wang, Guoqing; Qian, Minping; Gao, Huafang; Han, Bei; Chen, Yusheng; Hu, Yinghui; Geng, Rong; Hu, Chengping; Zhang, Wei; Yang, Jingping; Wan, Huanying; Yu, Qin; Wei, Liping; Li, Jiashu; Tian, Guizhen; Wang, Qiuyue; Hu, Ke; Wang, Siqin; Wang, Ruiqin; Du, Juan; He, Bei; Ma, Jianjun; Zhong, Xiaoning; Mu, Lan; Cai, Shaoxi; Zhu, Xiangdong; Xing, Wanli; Yu, Jun; Deng, Minghua; Gao, Zhancheng

2012-01-01

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship. Trial Registration ClinicalTrials.gov NCT00567827 PMID:22719933

Comparison of sampling methods for the detection of human rhinovirus RNA.

PubMed

Waris, Matti; Österback, Riikka; Lahti, Elina; Vuorinen, Tytti; Ruuskanen, Olli; Peltola, Ville

2013-09-01

Obtaining a nasal swab (NS) from a child for human rhinovirus (HRV) RNA detection is simple and well tolerated even for repeated sampling, but only few studies have compared them qualitatively and quantitatively with other sampling methods. Real-time PCR was used to study the stability of HRV genomes in swabs, and to compare different swabs and induced sputum specimens with nasopharyngeal aspirates (NPAs). Replicate swabs in a dry test tube were stored at room temperature or mailed to the laboratory before freezing, and compared to freshly frozen specimens. To compare sampling methods, paediatric patients had NPA, NS and throat swab collected. In paired sputum and NPA specimens, viral load was correlated to the amount of β-actin mRNA. Specimens were stable at room temperature for at least 4 days and survived mailing without loss of HRV detectability. As compared to NPA, NS had an equal diagnostic sensitivity, with no significant quantitative difference using flocked nylon swabs and a 2.2-fold drop in the average copy number using cotton swabs. The diagnostic sensitivity of cotton swab-collected throat specimens was 97%, with a 26-fold lower mean copy number. Sputum specimens had higher HRV RNA (2.3-fold) and β-actin mRNA (1.6-fold) copy numbers than NPAs, but there was a poor correlation between HRV RNA and β-actin mRNA. HRV remains well detectable by PCR in specimens mailed to the laboratory. The diagnostic efficacy of NPA can be obtained with NS, quantitative comparison and patient comfort favouring flocked nylon-tipped over cotton-tipped swabs. Copyright © 2013 Elsevier B.V. All rights reserved.

Frequency of tuberculous and non-tuberculous mycobacteria in HIV infected patients from Bogota, Colombia

PubMed Central

Murcia-Aranguren, Martha I; Gómez-Marin, Jorge E; Alvarado, Fernando S; Bustillo, José G; de Mendivelson, Ellen; Gómez, Bertha; León, Clara I; Triana, William A; Vargas, Erwing A; Rodríguez, Edgar

2001-01-01

Background The prevalence of infections by Mycobacterium tuberculosis and non-tuberculous Mycobacterium species in the HIV-infected patient population in Colombia was uncertain despite some pilot studies. We determined the frequency of isolation of Mycobacterium tuberculosis and of non-tuberculous Mycobacterium species in diverse body fluids of HIV-infected patients in Bogota, Colombia. Methods Patients who attended the three major HIV/AIDS healthcare centres in Bogota were prospectively studied over a six month period. A total of 286 patients were enrolled, 20% of them were hospitalized at some point during the study. Sixty four percent (64%) were classified as stage C, 25% as stage B, and 11% as stage A (CDC staging system, 1993). A total of 1,622 clinical samples (mostly paired samples of blood, sputum, stool, and urine) were processed for acid-fast bacilli (AFB) stain and culture. Results Overall 43 of 1,622 cultures (2.6%) were positive for mycobacteria. Twenty-two sputum samples were positive. Four patients were diagnosed with M. tuberculosis (1.4%). All isolates of M. tuberculosis were sensitive to common anti-tuberculous drugs. M. avium was isolated in thirteen patients (4.5%), but only in three of them the cultures originated from blood. The other isolates were obtained from stool, urine or sputum samples. In three cases, direct AFB smears of blood were positive. Two patients presented simultaneously with M. tuberculosis and M. avium. Conclusions Non-tuberculous Mycobacterium infections are frequent in HIV infected patients in Bogota. The diagnostic sensitivity for infection with tuberculous and non-tuberculous mycobacteria can be increased when diverse body fluids are processed from each patient. PMID:11722797

Ampicillin levels in sputum, serum, and saliva

PubMed Central

Stewart, Sheila M.; Fisher, Mary; Young, Joy E.; Lutz, W.

1970-01-01

The ampicillin levels in sputum, serum, and saliva from 40 patients receiving a dose of 250 mg., 26 patients receiving a dose of 500 mg., and 11 patients receiving a dose of 1 g. were estimated. The ampicillin was given orally four times daily. The 1-2 hour and 2-3 hour sputum levels were similar in individual patients. There was no difference in the range or mean sputum or saliva levels between specimens from patients receiving 250 mg. and 500 mg., but the levels were significantly higher after the 1 g. dose. The mean serum level showed a small increase after 500 mg. ampicillin as compared with the 250 mg. dose and a big increase after the 1 g. dose: only the latter difference was significant. The sputum levels were approximately 30 to 40 times lower than the corresponding serum levels. There was considerable scatter in the sputum level for any level of ampicillin in the serum: in only two of the 1-2 hour sputum specimens was there no detectable ampicillin. There was no correlation between the sputum levels and either the body weight or the dose in milligrams per kilogram. There was no evidence that corticosteroids or diuretics affected the sputum level. It was not possible to demonstrate any relationship between the purulence of the sputum and the level of ampicillin after doses of 250 mg. or 500 mg., but higher levels were found in the more purulent specimens after 1 g. doses. PMID:4318047

Evaluation of sodium hydroxide-N-acetyl-l-cysteine and 0.7% chlorhexidine decontamination methods for recovering Mycobacterium tuberculosis from sputum samples: A comparative analysis (The Gambia Experience).

PubMed

Gitteh, Ensa; Kweku Otu, Jacob; Jobarteh, Tijan; Mendy, Francis; Faal-Jawara, Isatou Tutty; Ofori-Anyinam, Nana Boatema; Ayorinde, Abigail; Secka, Ousman; Gehre, Florian

2016-12-01

To determine the culture yield and time to detection of mycobacterial growth between samples decontaminated using 0.7% chlorhexidine and sodium hydroxide-N-acetyl-l-cysteine (NaOH-NALC) and cultured on the Löwenstein-Jensen (LJ) medium. We also aimed to determine the contamination rate between the 0.7% chlorhexidine and NaOH-NALC decontamination methods. The study was carried out on 68 sputa samples (42 smear positives and 26 smear negatives). Of these 68 samples, 46 were collected from men and 26 from women with an approximate average age of 27years. All the sputum samples were decontaminated using the standard NaOH-NALC and 0.7% chlorhexidine methods. The concentrates were cultured in parallel on LJ media in which reading of the slope for mycobacterial growth was obtained daily for the first 2weeks and then weekly until week 8. The mycobacterial recovery rate, time to detection, and contamination rate were then compared. The overall recovery rate of mycobacterial growth on samples treated with both decontamination methods inoculated on LJ media is 51.5% (35/68). Specifically, mycobacterial growth rates on samples treated with 0.7% chlorhexidine and standard NaOH-NALC on LJ media were 61.8% (42/68) and 54.4% (37/68), respectively. However, the growth of Mycobacterium tuberculosis complex was faster on samples treated with 0.7% chlorhexidine than those treated with NaOH-NALC (average, 32±5days vs. 33±5.2days, respectively). The contamination rate on samples treated with 0.7% chlorhexidine was 1.5% (1/68), whereas on those treated with NaOH-NALC, the rate was 4.4% (3/68). The 0.7% chlorhexidine decontamination method is rapid and has less contamination rate in terms of mycobacterial recovery compared with the standard NaOH-NALC method. Therefore, the 0.7% chlorhexidine decontamination method would be an ideal alternative option for decontamination of sputum samples and recovery/isolation of M. tuberculosis in resource-poor countries. Copyright © 2016.

Significance of pregnancy test false negative results due to elevated levels of β-core fragment hCG.

PubMed

Johnson, Sarah; Eapen, Saji; Smith, Peter; Warren, Graham; Zinaman, Michael

2017-01-01

Very high levels of β-core fragment human chorionic gonadotrophin (βcf-hCG) are reported to potentially cause false negative results in point-of-care (POC)/over-the-counter (OTC) pregnancy tests. To investigate this further, women's daily early morning urine samples, collected prior to conception and during pregnancy, were analysed for intact, free β-, and βcf-hCG. The proportion of βcf-hCG was found to be related to that of hCG produced and in circulation. Therefore, best practice for accuracy testing of POC/OTC pregnancy tests would be to test devices against clinical samples containing high levels of βcf-hCG as well as standards spiked with biologically relevant ratios.

Clinical significance of plasma cell-free DNA mutations in PIK3CA, AKT1, and ESR1 gene according to treatment lines in ER-positive breast cancer.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Tomiguchi, Mai; Sueta, Aiko; Murakami, Keiichi; Iwase, Hirotaka

2018-02-26

The somatic activation of PI3K/AKT pathway mutations, PIK3CA and AKT1, and ESR1 mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive procedure to quickly assess and monitor disease progression or therapeutic effect in breast cancer (BC) patients, but the clinical significance of these mutations in late treatment lines (TLs) remains unclear. The subjects of this study were a total of 251 plasma samples from 128 estrogen receptor-positive (ER+) BC patients. Of these plasma samples, 133 were from 73 primary BC (PBC) patients, and 118 plasma samples were from 68 metastatic BC (MBC) patients. We developed droplet digital PCR (ddPCR) assays to verify the clinical significance of PIK3CA, AKT1, and ESR1 mutations in these patients. cfDNA PIK3CA mutations were observed in 15.1% of the PBC patients, while a cfDNA AKT1 mutation was observed in 1.4% of patients, and cfDNA ESR1 mutations were observed in 2.7% of patients. Patients with detectable cfDNA PIK3CA mutations were not associated with clinical outcomes. According to the TL, the prevalence of the PIK3CA and ESR1 mutations in cfDNA were lower in early TLs compared with late TLs. In the early TL group, patients with cfDNA PIK3CA mutations had a shorter time to treatment failure (TTF) than patients without mutations (P = 0.035). However, there was no statistically significant difference between patients with or without cfDNA ESR1 mutations. However, in the late TL group, patients with cfDNA ESR1 mutations had a shorter TTF than patients without mutations (P = 0.048). However, there was no statistically significant difference between patients with or without cfDNA PIK3CA mutations. Since the prevalence of cfDNA AKT1 mutation is low in both PBC and MBC patients, the impact of AKT1 mutations on the prognosis remains unclear. We have demonstrated the difference in the clinical significance of the hotspot PIK3CA, AKT1, and ESR1 mutations in cfDNA for each TL in ER+ BC patients.

Intervening to Improve Compassion Fatigue Resiliency in Forensic Nurses.

PubMed

Flarity, Kathleen; Nash, Kim; Jones, Whitney; Steinbruner, Dave

2016-01-01

Evidence suggests that forensic nurses (FNs) may be predisposed to compassion fatigue (CF) as a result of the challenges of the profession including high caseloads, role ambiguity, prosecution goals, vicarious traumatization, attending to survivors' emotional needs, and empowering victims. The purpose of this exploratory study was to determine the prevalence of CF in a convenience sample of 55 FNs and examine the treatment effectiveness of a multifaceted education program in an intervention subset. The intervention was intended to increase compassion satisfaction (CS) and decrease CF symptoms in FNs in one organization who participated in the training. The hypothesis was that the FNs will have moderate to high CF and moderate to high CS and the CF intervention will add in CF prevention and resiliency as measured with an improvement in the Professional Quality of Life test scores (B. H. ). In the prevalence sample, 69% of the FNs had moderate to low CS, 73% had moderate to high burnout, and 73% had moderate to high levels of secondary traumatic stress (STS). In the intervention group, the education program resulted in a statistically significant increase in CS and decreases in STS symptoms.

Concurrent Cryptococcal and Pneumocystis Pneumonia along with Pulmonary Tuberculosis in an HIV-Positive Patient: Lessons Learned for Early Management.

PubMed

Kaur, Ravinder; Gautam, Hitender; Maheshwari, Megha; Goyal, Ritu; Bhalla, Preena; Dewan, Richa

2011-01-01

We are presenting a 50-year-old patient of pulmonary tuberculosis, on anti-tuberculosis therapy (ATT) for last 2 months who presented with fever, cough, breathlessness, anorexia, and weight loss. The case was found to be HIV reactive. His sputum sample showed Candida albicans and Pneumocystis jirovecii. Fluconazole and cotrimoxazole + sulphamethoxazole were added. The index case did not respond to the treatment and his clinical condition started to deteriorate and he developed headache, vomiting, and dysphagia. Repeat sputum sample and cerebrospinal fluid (CSF) showed Cryptococcus neoformans which was found to be sensitive to Amphotericin B. Amphotericin B was added to the treatment and patient clinically responded to treatment. In conclusion, emphasis should be given to correct etiological identification, allowing appropriate treatment and decreasing the morbidity and mortality in these patients as concomitant opportunistic infections may cause diagnostic problems.

CORK Study in Cystic Fibrosis: Sustained Improvements in Ultra-Low-Dose Chest CT Scores After CFTR Modulation With Ivacaftor.

PubMed

Ronan, Nicola J; Einarsson, Gisli G; Twomey, Maria; Mooney, Denver; Mullane, David; NiChroinin, Muireann; O'Callaghan, Grace; Shanahan, Fergus; Murphy, Desmond M; O'Connor, Owen J; Shortt, Cathy A; Tunney, Michael M; Eustace, Joseph A; Maher, Michael M; Elborn, J Stuart; Plant, Barry J

2018-02-01

Ivacaftor produces significant clinical benefit in patients with cystic fibrosis (CF) with the G551D mutation. Prevalence of this mutation at the Cork CF Centre is 23%. This study assessed the impact of cystic fibrosis transmembrane conductance regulator modulation on multiple modalities of patient assessment. Thirty-three patients with the G551D mutation were assessed at baseline and prospectively every 3 months for 1 year after initiation of ivacaftor. Change in ultra-low-dose chest CT scans, blood inflammatory mediators, and the sputum microbiome were assessed. Significant improvements in FEV 1 , BMI, and sweat chloride levels were observed post-ivacaftor treatment. Improvement in ultra-low-dose CT imaging scores were observed after treatment, with significant mean reductions in total Bhalla score (P < .01), peribronchial thickening (P = .035), and extent of mucous plugging (P < .001). Reductions in circulating inflammatory markers, including interleukin (IL)-1β, IL-6, and IL-8 were demonstrated. There was a 30% reduction in the relative abundance of Pseudomonas species and an increase in the relative abundance of bacteria associated with more stable community structures. Posttreatment community richness increased significantly (P = .03). Early and sustained improvements on ultra-low-dose CT scores suggest it may be a useful method of evaluating treatment response. It paralleled improvement in symptoms, circulating inflammatory markers, and changes in the lung microbiota. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

Allergic Bronchopulmonary Aspergillosis: A Perplexing Clinical Entity

PubMed Central

Panjabi, Chandramani

2016-01-01

In susceptible individuals, inhalation of Aspergillus spores can affect the respiratory tract in many ways. These spores get trapped in the viscid sputum of asthmatic subjects which triggers a cascade of inflammatory reactions that can result in Aspergillus-induced asthma, allergic bronchopulmonary aspergillosis (ABPA), and allergic Aspergillus sinusitis (AAS). An immunologically mediated disease, ABPA, occurs predominantly in patients with asthma and cystic fibrosis (CF). A set of criteria, which is still evolving, is required for diagnosis. Imaging plays a compelling role in the diagnosis and monitoring of the disease. Demonstration of central bronchiectasis with normal tapering bronchi is still considered pathognomonic in patients without CF. Elevated serum IgE levels and Aspergillus-specific IgE and/or IgG are also vital for the diagnosis. Mucoid impaction occurring in the paranasal sinuses results in AAS, which also requires a set of diagnostic criteria. Demonstration of fungal elements in sinus material is the hallmark of AAS. In spite of similar histopathologic features, co-existence of ABPA and AAS is still uncommon. Oral corticosteroids continue to be the mainstay of management of allergic aspergillosis. Antifungal agents play an adjunctive role in ABPA as they help reduce the fungal load. Saprophytic colonization in cavitary ABPA may lead to aspergilloma formation, which could increase the severity of the disease. The presence of ABPA, AAS, and aspergilloma in the same patient has also been documented. All patients with Aspergillus-sensitized asthma must be screened for ABPA, and AAS should always be looked for. PMID:27126721

Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

PubMed

Sonnleitner, Elisabeth; Valentini, Martina; Wenner, Nicolas; Haichar, Feth el Zahar; Haas, Dieter; Lapouge, Karine

2012-01-01

The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

Omega-3 fatty acids (from fish oils) for cystic fibrosis.

PubMed

McKarney, C; Everard, M; N'Diaye, T

2007-10-17

Epidemiological and other studies suggest that a diet rich in omega-3 essential fatty acids (derived from fish oil) may have beneficial anti-inflammatory effects for chronic conditions such as cystic fibrosis (CF). To determine whether there is evidence that omega-3 polyunsaturated fatty acid supplementation reduces morbidity and mortality. To identify any adverse events associated with omega-3 polyunsaturated fatty acid supplementation. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. Authors and persons interested in the subject of the review were contacted. Most recent search: April 2007 Randomised controlled trials in people with CF comparing omega-3 fatty acid supplements with placebo. Two authors independently selected the studies for inclusion in the review, independently extracted data and assessed the methodological quality of the studies. Searches identified seven studies; three of which, involving 48 participants, were eligible for inclusion in the review. Two studies compared omega-3 fatty acids to olive oil controls for a six-week treatment period. One study compared omega-3 fatty acids in the form of a liquid dietary supplement containing polyunsaturated fatty acids to a liquid dietary supplement control for six months. One short-term study (19 participants) comparing omega-3 to placebo reported a significant improvement in forced expiratory volume in one second, forced vital capacity and Shwachman score and a reduction in sputum volume in the omega-3 group. The longer-term study (17 participants) demonstrated a significant increase in essential fatty acid content in neutrophil membranes in study participants taking omega-3 supplements compared to placebo, weighted mean difference 0.90 (95% confidence interval 0.46 to 1.34). This review found that regular omega-3 supplements may provide some benefits for people with CF with relatively few adverse effects, although the evidence is insufficient to draw firm conclusions or to recommend routine use of supplements of omega-3 fatty acids in people with CF. This review has highlighted the lack of data for many of the outcomes likely to be meaningful to people with or making treatment decisions about CF. A large, long-term, multicentre, randomised controlled study is needed in order to determine if there is a significant therapeutic effect and to assess the influence of disease severity, dosage and duration of treatment. Future researchers should note the need for additional pancreatic enzymes.

Size Effect on the Mechanical Properties of CF Winding Composite

NASA Astrophysics Data System (ADS)

Cui, Yuqing; Yin, Zhongwei

2017-12-01

Mechanical properties of filament winding composites are usually tested by NOL ring samples. Few people have studied the size effect of winding composite samples on the testing result of mechanical property. In this research, winding composite thickness, diameter, and geometry of NOL ring samples were prepared to investigate the size effect on the mechanical strength of carbon fiber (CF) winding composite. The CF T700, T1000, M40, and M50 were adopted for the winding composite, while the matrix was epoxy resin. Test results show that the tensile strength and ILSS of composites decreases monotonically with an increase of thickness from 1 mm to 4 mm. The mechanical strength of composite samples increases monotonically with the increase in diameter from 100 mm to 189 mm. The mechanical strength of composite samples with two flat sides are higher than those of cyclic annular samples.

Identification of Burkholderia spp. in the Clinical Microbiology Laboratory: Comparison of Conventional and Molecular Methods

PubMed Central

van Pelt, Cindy; Verduin, Cees M.; Goessens, Wil H. F.; Vos, Margreet C.; Tümmler, Burkhard; Segonds, Christine; Reubsaet, Frans; Verbrugh, Henri; van Belkum, Alex

1999-01-01

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended. PMID:10364579

Assessment of Aspergillus sensitization or persistent carriage as a factor in lung function impairment in cystic fibrosis patients.

PubMed

Fillaux, Judith; Brémont, François; Murris, Marléne; Cassaing, Sophie; Rittié, Jean-Luc; Tétu, Laurent; Segonds, Christine; Abbal, Michel; Bieth, Eric; Berry, Antoine; Pipy, Bernard; Magnaval, Jean-François

2012-11-01

Cystic fibrosis (CF) patients presenting with persistent carriage of, or sensitization to, Aspergillus fumigatus are often treated with antifungal therapies because the presence of the fungus is commonly thought to impede lung function, even in the absence of allergic bronchopulmonary aspergillosis (ABPA). The aim of this study was to assess Aspergillus-related status modulating the forced expiratory volume in 1 s (FEV₁) of CF patients. From 1995 to 2007, 251 patients were evaluated. Demographic data, cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations, body mass index, and FEV(1) were recorded. The presence of A. fumigatus and Pseudomonas aeruginosa in sputum and the levels of A. fumigatus precipitin, total IgE (t-IgE), and specific anti-A. fumigatus IgE (Af-IgE) were determined. Patients were divided into 3 groups: (1) ABPA: A. fumigatus precipitin ≥3 lines, Af-IgE > 0.35 IU/ml, and t-IgE ≥500 IU/ml; (2) sensitization: Af-IgE > 0.35 IU/ml but t-IgE < 500 IU/ml; and (3) persistent carriage: Af-IgE ≤ 0.35 IU/ml with either an A. fumigatus persistent positive culture or an A. fumigatus precipitin ≥3 lines, provided this serological finding had been found associated with at least 1 A. fumigatus-positive culture. The remaining patients represented the control group. A multivariate analysis was carried out with FEV(1) as the outcome variable. ABPA, sensitization, and persistent carriage were significantly associated with a larger decline in FEV₁ compared with the control group, with odds ratios of 15.9, 14.9, and 10.7, respectively. This association was independent of other associated factors (P. aeruginosa transient detection, age, being underweight, and low FEV₁ at baseline). In addition to ABPA, sensitization and persistent carriage appear to have an impact on pulmonary function in CF patients.

Under-reporting of sputum smear-positive tuberculosis cases in Kenya.

PubMed

Tollefson, D; Ngari, F; Mwakala, M; Gethi, D; Kipruto, H; Cain, K; Bloss, E

2016-10-01

Although an estimated three million tuberculosis (TB) cases worldwide are missed by national TB programs annually, the level of under-reporting of diagnosed cases in high TB burden settings is largely unknown. To quantify and describe under-reporting of sputum smear-positive TB cases in Kenya. A national-level retrospective TB inventory study was conducted. All sputum smear-positive TB cases diagnosed by public or private laboratories during 1 April-30 June 2013 were extracted from laboratory registers in 73 randomly sampled subcounties and matched to TB cases in the national TB surveillance system (TIBU). Bivariate and multivariate analyses were conducted. In the subcounties sampled, 715 of 3409 smear-positive TB cases in laboratory registers were not found in TIBU. The estimated level of under-reporting of smear-positive TB cases in Kenya was 20.7% (95%CI 18.4-23.0). Under-reporting was greatest in subcounties with a high TB burden. Unreported cases were more likely to be patients aged ⩾55 years, have scanty smear results, and be diagnosed at large facilities, private facilities, and facilities in high TB burden regions. In Kenya, one fifth of smear-positive TB cases diagnosed during the study period went unreported, suggesting that the true TB burden is higher than reported. TB surveillance in Kenya should be strengthened to ensure all diagnosed TB cases are reported.

Reemergence of Lower-Airway Microbiota in Lung Transplant Patients with Cystic Fibrosis.

PubMed

Syed, Saad A; Whelan, Fiona J; Waddell, Barbara; Rabin, Harvey R; Parkins, Michael D; Surette, Michael G

2016-12-01

Chronic lung infections are a hallmark of cystic fibrosis; they are responsible for progressive airway destruction and ultimately lead to respiratory death or the requirement for life-saving bilateral lung transplant. Furthermore, recurrent isolation of airway pathogens such as Pseudomonas aeruginosa in the allograft after transplant is associated with adverse outcomes, including bronchiolitis obliterans syndrome and acute infections. Little information exists on the impact of bilateral lung transplant on the lower-airway microbiota. To compare, at a microbiome and single-pathogen level (P. aeruginosa), the bacterial communities in pre- and post-transplant samples. We retrospectively accessed our biobank of sputum samples and sputum-derived bacterial pathogens for patients who had matched samples, including those who were clinically stable before transplant, those who had a pulmonary exacerbation before transplant, and those who had pulmonary exacerbation after transplant. We used 16S ribosomal RNA gene sequencing to characterize the lower-airway microbiome of 14 adult transplant patients with cystic fibrosis. Genotyping and phenotyping of P. aeruginosa isolates from 12 of these patients with matched isolates was performed. Although α-diversity (richness and evenness) of patient microbiomes was similar before and after transplant, β- diversity (core microbiome composition) measures stratified patients evenly into two groups with more similar and more dissimilar communities. P. aeruginosa strains isolated before transplant were found to reemerge in 11 of 12 patients; however, phenotypic variation was observed. These findings indicate that recolonization by P. aeruginosa after transplant is almost always strain specific, suggesting a within-host source. The polymicrobial colonization of the airways after transplant does not always reflect the pretransplant sputum microbiota.

Profiling over 1500 lipids in induced lung sputum and the implications in studying lung diseases.

PubMed

t'Kindt, Ruben; Telenga, Eef D; Jorge, Lucie; Van Oosterhout, Antoon J M; Sandra, Pat; Ten Hacken, Nick H T; Sandra, Koen

2015-01-01

Induced lung sputum is a valuable matrix in the study of respiratory diseases. Although the methodology of sputum collection has evolved to a point where it is repeatable and responsive to inflammation, its use in molecular profiling studies is still limited. Here, an in-depth lipid profiling of induced lung sputum using high-resolution liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) is described. An enormous complexity in lipid composition could be revealed. Over 1500 intact lipids, originating from 6 major lipid classes, have been accurately identified in 120 μL of induced sputum. By number and measured intensity, glycerophospholipids represent the largest lipid class, followed by sphingolipids, glycerolipids, fatty acyls, sterol lipids, and prenol lipids. Several prenol lipids, originating from tobacco, could be detected in the lung sputum of smokers. To illustrate the utility of the methodology in studying respiratory diseases, a comparative lipid screening was performed on lung sputum extracts in order to study the effect of Chronic Obstructive Pulmonary Disease (COPD) on the lung barrier lipidome. Results show that sphingolipid expression in induced sputum significantly differs between smokers with and without COPD.

Sputum Inflammatory Mediators Are Increased in Aspergillus fumigatus Culture-Positive Asthmatics

PubMed Central

Ghebre, Michael A; Desai, Dhananjay; Singapuri, Amisha; Woods, Joanne; Rapley, Laura; Cohen, Suzanne; Herath, Athula; Wardlaw, Andrew J; Pashley, Catherine H; May, Richard

2017-01-01

Aspergillus fumigatus sensitization and culture in asthma are associated with disease severity and lung function impairment, but their relationship with airway inflammation is poorly understood. We investigated the profile of 24 sputum inflammatory mediators in A. fumigatus culture-positive or-negative moderate-to-severe asthmatics. Fifty-two subjects were recruited from a single center. A. fumigatus was cultured from 19 asthmatics. Asthma control, symptom score, lung function, and sputum cell count were not significantly different between the asthmatics with and without a positive A. fumigatus culture. All of the sputum mediators were numerically increased in subjects with a positive versus negative sputum A. fumigatus culture. Sputum TNF-R2 was significantly elevated (P=0.03) and the mediator that best distinguished A. fumigatus culture-positive from culture-negative subjects (receiver-operator characteristic area under the curve 0.66 [95% CI: 0.51 to 0.82, P=0.045]). A. fumigates-positive culture in moderate-to-severe asthma is associated with increased inflammatory sputum mediators. PMID:28102063

Cell-free DNA detected by "liquid biopsy" as a potential prognostic biomarker in early breast cancer.

PubMed

Maltoni, Roberta; Casadio, Valentina; Ravaioli, Sara; Foca, Flavia; Tumedei, Maria Maddalena; Salvi, Samanta; Martignano, Filippo; Calistri, Daniele; Rocca, Andrea; Schirone, Alessio; Amadori, Dino; Bravaccini, Sara

2017-03-07

As conventional biomarkers for defining breast cancer (BC) subtypes are not always capable of predicting prognosis, search for new biomarkers which can be easily detected by liquid biopsy is ongoing. It has long been known that cell-free DNA (CF-DNA) could be a promising diagnostic and prognostic marker in different tumor types, although its prognostic value in BC is yet to be confirmed. This retrospective study evaluated the prognostic role of CF-DNA quantity and integrity of HER2, MYC, BCAS1 and PI3KCA, which are frequently altered in BC. We collected 79 serum samples before surgery from women at first diagnosis of BC at Forlì Hospital (Italy) from 2002 to 2010. Twenty-one relapsed and 58 non-relapsed patients were matched by subtype and age. Blood samples were also collected from 10 healthy donors. All samples were analyzed by Real Time PCR for CF-DNA quantity and integrity of all oncogenes. Except for MYC, BC patients showed significantly higher median values of CF-DNA quantity (ng) than healthy controls, who had higher integrity and lower apoptotic index. A difference nearing statistical significance was observed for HER2 short CF-DNA (p = 0.078, AUC value: 0.6305). HER2 short CF-DNA showed an odds ratio of 1.39 for disease recurrence with p = 0.056 (95% CI 0.991-1.973). Our study suggests that CF-DNA detected as liquid biopsy could have great potential in clinical practice once demonstration of its clinical validity and utility has been provided by prospective studies with robust assays.

Fungal epidemiology and diversity in cystic fibrosis patients over a 5-year period in a national reference center.

PubMed

Ziesing, S; Suerbaum, S; Sedlacek, L

2016-11-01

The knowledge on prevalence rates of yeasts and moulds in patients with cystic fibrosis (CF) in Germany is scarce. The aim of this report is to give an overview of the diversity and epidemiology of fungal species in CF patients. Over a 5-year period, all fungal isolates cultured from microbiological specimen from CF patients were recorded. Beside standard bacteriological culture media two fungal media were used for cultivation. Species were identified by microscopy, biochemical profiling, MALDI-TOF analysis or DNA sequencing methods. In sum, 25,975 clinical samples from CF patients were analyzed. About 75% of CF patients were colonized by yeasts, mainly Candida albicans (38%) and Candida dubliniensis (12%). In 35% of the patients Aspergillus spp. (Aspergillus fumigatus: 29%) were detected, followed by Exophiala dermatitidis and Scedosporium/Lomentospora complex isolates (4% each). Data for other fungal species are shown. Over a 5-year period, the epidemiology of fungal species detected in CF patients was relatively constant. Clinical microbiology laboratories should carefully monitor samples from CF patients for newly occurring fungal pathogens. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluation of Digital PCR as a Technique for Monitoring Acute Rejection in Kidney Transplantation.

PubMed

Lee, Hyeseon; Park, Young-Mi; We, Yu-Mee; Han, Duck Jong; Seo, Jung-Woo; Moon, Haena; Lee, Yu-Ho; Kim, Yang-Gyun; Moon, Ju-Young; Lee, Sang-Ho; Lee, Jong-Keuk

2017-03-01

Early detection and proper management of kidney rejection are crucial for the long-term health of a transplant recipient. Recipients are normally monitored by serum creatinine measurement and sometimes with graft biopsies. Donor-derived cell-free deoxyribonucleic acid (cfDNA) in the recipient's plasma and/or urine may be a better indicator of acute rejection. We evaluated digital PCR (dPCR) as a system for monitoring graft status using single nucleotide polymorphism (SNP)-based detection of donor DNA in plasma or urine. We compared the detection abilities of the QX200, RainDrop, and QuantStudio 3D dPCR systems. The QX200 was the most accurate and sensitive. Plasma and/or urine samples were isolated from 34 kidney recipients at multiple time points after transplantation, and analyzed by dPCR using the QX200. We found that donor DNA was almost undetectable in plasma DNA samples, whereas a high percentage of donor DNA was measured in urine DNA samples, indicating that urine is a good source of cfDNA for patient monitoring. We found that at least 24% of the highly polymorphic SNPs used to identify individuals could also identify donor cfDNA in transplant patient samples. Our results further showed that autosomal, sex-specific, and mitochondrial SNPs were suitable markers for identifying donor cfDNA. Finally, we found that donor-derived cfDNA measurement by dPCR was not sufficient to predict a patient's clinical condition. Our results indicate that donor-derived cfDNA is not an accurate predictor of kidney status in kidney transplant patients.

Bacterial biogeography of adult airways in atopic asthma.

PubMed

Durack, Juliana; Huang, Yvonne J; Nariya, Snehal; Christian, Laura S; Mark Ansel, K; Beigelman, Avraham; Castro, Mario; Dyer, Anne-Marie; Israel, Elliot; Kraft, Monica; Martin, Richard J; Mauger, David T; Rosenberg, Sharon R; King, Tonya S; White, Steven R; Denlinger, Loren C; Holguin, Fernando; Lazarus, Stephen C; Lugogo, Njira; Peters, Stephen P; Smith, Lewis J; Wechsler, Michael E; Lynch, Susan V; Boushey, Homer A

2018-06-09

Perturbations to the composition and function of bronchial bacterial communities appear to contribute to the pathophysiology of asthma. Unraveling the nature and mechanisms of these complex associations will require large longitudinal studies, for which bronchoscopy is poorly suited. Studies of samples obtained by sputum induction and nasopharyngeal brushing or lavage have also reported asthma-associated microbiota characteristics. It remains unknown, however, whether the microbiota detected in these less-invasive sample types reflect the composition of bronchial microbiota in asthma. Bacterial microbiota in paired protected bronchial brushings (BB; n = 45), induced sputum (IS; n = 45), oral wash (OW; n = 45), and nasal brushings (NB; n = 27) from adults with mild atopic asthma (AA), atopy without asthma (ANA), and healthy controls (HC) were profiled using 16S rRNA gene sequencing. Though microbiota composition varied with sample type (p < 0.001), compositional similarity was greatest for BB-IS, particularly in AAs and ANAs. The abundance of genera detected in BB correlated with those detected in IS and OW (r median [IQR] 0.869 [0.748-0.942] and 0.822 [0.687-0.909] respectively), but not with those in NB (r = 0.004 [- 0.003-0.011]). The number of taxa shared between IS-BB and NB-BB was greater in AAs than in HCs (p < 0.05) and included taxa previously associated with asthma. Of the genera abundant in NB, only Moraxella correlated positively with abundance in BB; specific members of this genus were shared between the two compartments only in AAs. Relative abundance of Moraxella in NB of AAs correlated negatively with that of Corynebacterium but positively with markers of eosinophilic inflammation in the blood and BAL fluid. The genus, Corynebacterium, trended to dominate all NB samples of HCs but only half of AAs (p = 0.07), in whom abundance of this genus was negatively associated with markers of eosinophilic inflammation. Induced sputum is superior to nasal brush or oral wash for assessing bronchial microbiota composition in asthmatic adults. Although compositionally similar to the bronchial microbiota, the microbiota in induced sputum are distinct, reflecting enrichment of oral bacteria. Specific bacterial genera are shared between the nasal and the bronchial mucosa which are associated with markers of systemic and bronchial inflammation.

Organization of Patient Management and Fungal Epidemiology in Cystic Fibrosis.

PubMed

Schwarz, Carsten; Bouchara, Jean-Philippe; Buzina, Walter; Chrenkova, Vanda; Dmeńska, Hanna; de la Pedrosa, Elia Gomez Garcia; Cantón, Rafael; Fiscarelli, Ersilia; Le Govic, Yohann; Kondori, Nahid; Matos, Tadeja; Romanowska, Ewa; Ziesing, Stefan; Sedlacek, Ludwig

2018-02-01

The achievement of a better life for cystic fibrosis (CF) patients is mainly caused by a better management and infection control over the last three decades. Herein, we want to summarize the cornerstones for an effective management of CF patients and to give an overview of the knowledge about the fungal epidemiology in this clinical context in Europe. Data from a retrospective analysis encompassing 66,616 samples from 3235 CF patients followed-up in 9 CF centers from different European countries are shown.

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

2015-05-01

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

Impact of GeneXpert MTB/RIF assay on triage of respiratory isolation rooms for inpatients with presumed tuberculosis: a hypothetical trial.

PubMed

Chaisson, Lelia H; Roemer, Marguerite; Cantu, David; Haller, Barbara; Millman, Alexander J; Cattamanchi, Adithya; Davis, J Lucian

2014-11-15

Placing inpatients with presumed active pulmonary tuberculosis in respiratory isolation pending results of serial sputum acid-fast bacilli (AFB) smear microscopy is standard practice in high-income countries. However, this diagnostic strategy is slow and yields few tuberculosis diagnoses. We sought to determine if replacing microscopy with the GeneXpert MTB/RIF (Xpert) nucleic acid amplification assay could reduce testing time and usage of isolation rooms. We prospectively followed inpatients at San Francisco General Hospital undergoing tuberculosis evaluation. We performed smear microscopy and Xpert testing on concentrated sputum, and calculated diagnostic accuracy for both strategies in reference to serial sputum mycobacterial culture. We measured turnaround time for microscopy and estimated hypothetical turnaround times for Xpert on concentrated and unconcentrated sputum. We compared median and total isolation times for microscopy to those estimated for the 2 Xpert strategies. Among 139 patients with 142 admissions, median age was 54 years (interquartile range [IQR], 43-60 years); 32 (23%) patients were female, and 42 (30%) were HIV seropositive. Serial sputum smear microscopy and a single concentrated sputum Xpert had identical sensitivity (89%; 95% confidence interval [CI], 52%-100%) and similar specificity (99% [95% CI, 96%-100%] vs 100% [95% CI, 97%-100%]). A single concentrated sputum Xpert could have saved a median of 35 hours (IQR, 24-36 hours) in unnecessary isolation compared with microscopy, and a single unconcentrated sputum Xpert, 45 hours (IQR, 35-46 hours). Replacing serial sputum smear microscopy with a single sputum Xpert could eliminate most unnecessary isolation for inpatients with presumed tuberculosis, greatly benefiting patients and hospitals. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Sputum neutrophil counts are associated with more severe asthma phenotypes using cluster analysis.

PubMed

Moore, Wendy C; Hastie, Annette T; Li, Xingnan; Li, Huashi; Busse, William W; Jarjour, Nizar N; Wenzel, Sally E; Peters, Stephen P; Meyers, Deborah A; Bleecker, Eugene R

2014-06-01

Clinical cluster analysis from the Severe Asthma Research Program (SARP) identified 5 asthma subphenotypes that represent the severity spectrum of early-onset allergic asthma, late-onset severe asthma, and severe asthma with chronic obstructive pulmonary disease characteristics. Analysis of induced sputum from a subset of SARP subjects showed 4 sputum inflammatory cellular patterns. Subjects with concurrent increases in eosinophil (≥2%) and neutrophil (≥40%) percentages had characteristics of very severe asthma. To better understand interactions between inflammation and clinical subphenotypes, we integrated inflammatory cellular measures and clinical variables in a new cluster analysis. Participants in SARP who underwent sputum induction at 3 clinical sites were included in this analysis (n = 423). Fifteen variables, including clinical characteristics and blood and sputum inflammatory cell assessments, were selected using factor analysis for unsupervised cluster analysis. Four phenotypic clusters were identified. Cluster A (n = 132) and B (n = 127) subjects had mild-to-moderate early-onset allergic asthma with paucigranulocytic or eosinophilic sputum inflammatory cell patterns. In contrast, these inflammatory patterns were present in only 7% of cluster C (n = 117) and D (n = 47) subjects who had moderate-to-severe asthma with frequent health care use despite treatment with high doses of inhaled or oral corticosteroids and, in cluster D, reduced lung function. The majority of these subjects (>83%) had sputum neutrophilia either alone or with concurrent sputum eosinophilia. Baseline lung function and sputum neutrophil percentages were the most important variables determining cluster assignment. This multivariate approach identified 4 asthma subphenotypes representing the severity spectrum from mild-to-moderate allergic asthma with minimal or eosinophil-predominant sputum inflammation to moderate-to-severe asthma with neutrophil-predominant or mixed granulocytic inflammation. Published by Mosby, Inc.

Sputum neutrophils are associated with more severe asthma phenotypes using cluster analysis

PubMed Central

Moore, Wendy C.; Hastie, Annette T.; Li, Xingnan; Li, Huashi; Busse, William W.; Jarjour, Nizar N.; Wenzel, Sally E.; Peters, Stephen P.; Meyers, Deborah A.; Bleecker, Eugene R.

2013-01-01

Background Clinical cluster analysis from the Severe Asthma Research Program (SARP) identified five asthma subphenotypes that represent the severity spectrum of early onset allergic asthma, late onset severe asthma and severe asthma with COPD characteristics. Analysis of induced sputum from a subset of SARP subjects showed four sputum inflammatory cellular patterns. Subjects with concurrent increases in eosinophils (≥2%) and neutrophils (≥40%) had characteristics of very severe asthma. Objective To better understand interactions between inflammation and clinical subphenotypes we integrated inflammatory cellular measures and clinical variables in a new cluster analysis. Methods Participants in SARP at three clinical sites who underwent sputum induction were included in this analysis (n=423). Fifteen variables including clinical characteristics and blood and sputum inflammatory cell assessments were selected by factor analysis for unsupervised cluster analysis. Results Four phenotypic clusters were identified. Cluster A (n=132) and B (n=127) subjects had mild-moderate early onset allergic asthma with paucigranulocytic or eosinophilic sputum inflammatory cell patterns. In contrast, these inflammatory patterns were present in only 7% of Cluster C (n=117) and D (n=47) subjects who had moderate-severe asthma with frequent health care utilization despite treatment with high doses of inhaled or oral corticosteroids, and in Cluster D, reduced lung function. The majority these subjects (>83%) had sputum neutrophilia either alone or with concurrent sputum eosinophilia. Baseline lung function and sputum neutrophils were the most important variables determining cluster assignment. Conclusion This multivariate approach identified four asthma subphenotypes representing the severity spectrum from mild-moderate allergic asthma with minimal or eosinophilic predominant sputum inflammation to moderate-severe asthma with neutrophilic predominant or mixed granulocytic inflammation. PMID:24332216

Asthma Control and Sputum Eosinophils: A Longitudinal Study in Daily Practice.

PubMed

Demarche, Sophie F; Schleich, Florence N; Paulus, Virginie A; Henket, Monique A; Van Hees, Thierry J; Louis, Renaud E

Longitudinal trials have suggested that asthma control may be influenced by fluctuations in eosinophilic inflammation. This association has however never been confirmed in daily practice. To investigate the relationship between asthma control and sputum eosinophils in clinical practice. A retrospective longitudinal study was conducted on 187 patients with asthma with at least 2 successful sputum inductions at our Asthma Clinic. Linear mixed models were used to assess the relationship between asthma control and individual changes in sputum eosinophils. Receiver-operating characteristic curves were constructed to define minimal important differences (MIDs) of sputum eosinophils associated with a change of at least 0.5 in Asthma Control Questionnaire (ACQ) score. Then, a validation cohort of 79 patients with asthma was recruited to reassess this relationship and the accuracy of the MID values. A multivariate analysis showed that asthma control was independently associated with individual fluctuations in sputum eosinophil count (P < .001). In patients with intermittent/persistently eosinophilic asthma, we calculated a minimal important decrease of 4.3% in the percentage of sputum eosinophils (area under the curve [AUC], 0.69; P < .001) or 3.4-fold (AUC, 0.65; P = .003) for a significant improvement in asthma control and a minimal important increase of 3.5% (AUC, 0.67; P = .004) or 1.8-fold (AUC, 0.63; P = .02) for a significant worsening in asthma control. The association between asthma control and sputum eosinophils and the accuracy of the MIDs of sputum eosinophils were confirmed in the validation cohort. At the individual level, asthma control was associated with fluctuations in sputum eosinophil count over time. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

False-Positive Gen-Probe Direct Mycobacterium tuberculosis Amplification Test Results for Patients with Pulmonary M. kansasii and M. avium Infections

PubMed Central

Jorgensen, James H.; Salinas, Jesse R.; Paxson, Rosemary; Magnon, Karen; Patterson, Jan E.; Patterson, Thomas F.

1999-01-01

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test has been approved for use in the United States for the rapid diagnosis of pulmonary tuberculosis in patients with acid-fast smear-positive sputum samples since 1996. Four patients infected with human immunodeficiency virus and one chronic pulmonary-disease patient seen in our institutions with abnormal chest radiographs and fluorochrome stain-positive sputa were evaluated for tuberculosis, including performance of the MTD test on expectorated sputum samples. Three of these five patients’ sputa were highly smear-positive (i.e., more than 100 bacilli per high-power field), while two patient’s sputa contained 1 to 10 bacilli per field. MTD results on sputum specimens from these patients ranged from 43,498 to 193,858 relative light units (RLU). Gen-Probe has defined values of at least 30,000 RLU as indicative of a positive test, i.e., the presence of Mycobacterium tuberculosis RNA. Four of the patients’ sputum cultures yielded growth of M. kansasii within 6 to 12 days, and the fifth produced growth of M. avium only. One patient’s culture contained both M. kansasii and M. avium, but none of the initial or follow-up cultures from these five patients revealed M. tuberculosis. However, subsequent cultures from three of the patients again revealed M. kansasii. During the period of this study, in which MTD tests were performed on smear-positive sputum specimens from 82 patients, four of seven patients with culture-proven M. kansasii pulmonary infections yielded one or more false-positive MTD tests. The MTD sensitivity observed in this study was 93.8%, and the specificity was 85.3%. Five cultures of M. kansasii (including three of these patients’ isolates and M. kansasii ATCC 12478), and cultures of several other species were examined at densities of 105 to 107 viable CFU/ml by the MTD test. All five isolates of M. kansasii and three of three isolates of M. simiae yielded false-positive test results, with readings of 75,191 to 335,591 RLU. These findings indicate that low-level false-positive MTD results can occur due to the presence of M. kansasii, M. avium, and possibly other Mycobacterium species other than M. tuberculosis in sputum. Low-level positive MTD results of 30,000 to 500,000 RLU should be interpreted in light of these findings. It remains to be determined if the enhanced MTD test (MTD 2) recently released by Gen-Probe will provide greater specificity than that observed in this report with its first-generation test. PMID:9854086

Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients.

PubMed

Koeppel, Florence; Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

2017-01-01

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients

PubMed Central

Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

2017-01-01

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation. PMID:29161279

Predictive value of anaemia for tuberculosis in HIV-infected patients in sub-Saharan Africa: an indication for routine microbiological investigation using new rapid assays

PubMed Central

Kerkhoff, Andrew D.; Wood, Robin; Vogt, Monica; Lawn, Stephen D.

2014-01-01

Background The relationship between anaemia and undiagnosed tuberculosis (TB) in patients living with HIV in sub-Saharan Africa is incompletely defined. We assessed the prevalence of TB among those with HIV-related anaemia and evaluated new means of rapid TB diagnosis. Methodology Blood haemoglobin levels were measured in unselected antiretroviral treatment (ART)-naïve patients in Cape Town, South Africa, and anaemia was classified according to WHO criteria. All patients were screened for TB by testing paired sputum samples using liquid culture (reference standard), fluorescence microscopy and Xpert MTB/RIF. Urine samples were tested for lipoarabinomannan (LAM) using the Determine TB-LAM diagnostic assay. Results Of 602 adults screened, 485 had complete results. Normal haemoglobin levels were found in 44.5% (n=216) of patients and mild, moderate or severe anaemia were present in 24.9% (n=121), 25.4% (n=123) and 5.2% (n=25) of patients, respectively. Culture-confirmed pulmonary TB was diagnosed in 8.8% (19/216) of those without anaemia compared to 16.5% (20/121), 26.0% (32/123) and 40.0% (10/25) with mild, moderate or severe anaemia, respectively (p<0.001). Anaemia was a strong independent predictor of TB. The sensitivities of diagnostic assays were much higher among those with moderate/severe anaemia compared to those with no/mild anaemia using sputum microscopy (42.9% vs 15.4%); urine LAM (54.8% vs 0%); sputum microscopy plus urine LAM (71.4% vs 15.4%) and sputum Xpert (73.8% vs 41.0%) (P<0.01 for all). Conclusions A very high prevalence of undiagnosed TB was found in patients with moderate or severe anaemia. Such patients should be prioritized for routine microbiological investigation using rapid diagnostic assays. PMID:24346639

Curing Effects on Interfacial Adhesion between Recycled Carbon Fiber and Epoxy Resin Heated by Microwave Irradiation

PubMed Central

Shimamoto, Daisuke; Hotta, Yuji

2018-01-01

The interfacial adhesion of recycled carbon fiber (CF) reinforced epoxy composite heated by microwave (MW) irradiation were investigated by changing the curing state of the epoxy resin. The recycled CF was recovered from the composite, which was prepared by vacuum-assisted resin transfer molding, by thermal degradation at 500 or 600 °C. Thermogravimetric analysis showed that the heating at 600 °C caused rough damage to the CF surface, whereas recycled CF recovered at 500 °C have few defects. The interfacial shear strength (IFSS) between recycled CF and epoxy resin was measured by a single-fiber fragmentation test. The test specimen was heated by MW after mixing the epoxy resin with a curing agent or pre-curing, in order to investigate the curing effects on the matrix resin. The IFSSs of the MW-irradiated samples were significantly varied by the curing state of the epoxy resin and the surface condition of recycled CF, resulting that they were 99.5 to 131.7% of oven heated samples Furthermore, rheological measurements showed that the viscosity and shrinking behaviors of epoxy resin were affected based on the curing state of epoxy resin before MW irradiation. PMID:29587422

Curing Effects on Interfacial Adhesion between Recycled Carbon Fiber and Epoxy Resin Heated by Microwave Irradiation.

PubMed

Tominaga, Yuichi; Shimamoto, Daisuke; Hotta, Yuji

2018-03-26

The interfacial adhesion of recycled carbon fiber (CF) reinforced epoxy composite heated by microwave (MW) irradiation were investigated by changing the curing state of the epoxy resin. The recycled CF was recovered from the composite, which was prepared by vacuum-assisted resin transfer molding, by thermal degradation at 500 or 600 °C. Thermogravimetric analysis showed that the heating at 600 °C caused rough damage to the CF surface, whereas recycled CF recovered at 500 °C have few defects. The interfacial shear strength (IFSS) between recycled CF and epoxy resin was measured by a single-fiber fragmentation test. The test specimen was heated by MW after mixing the epoxy resin with a curing agent or pre-curing, in order to investigate the curing effects on the matrix resin. The IFSSs of the MW-irradiated samples were significantly varied by the curing state of the epoxy resin and the surface condition of recycled CF, resulting that they were 99.5 to 131.7% of oven heated samples Furthermore, rheological measurements showed that the viscosity and shrinking behaviors of epoxy resin were affected based on the curing state of epoxy resin before MW irradiation.

Association of sputum and blood eosinophil concentrations with clinical measures of COPD severity: an analysis of the SPIROMICS cohort.

PubMed

Hastie, Annette T; Martinez, Fernando J; Curtis, Jeffrey L; Doerschuk, Claire M; Hansel, Nadia N; Christenson, Stephanie; Putcha, Nirupama; Ortega, Victor E; Li, Xingnan; Barr, R Graham; Carretta, Elizabeth E; Couper, David J; Cooper, Christopher B; Hoffman, Eric A; Kanner, Richard E; Kleerup, Eric; O'Neal, Wanda K; Paine, Richard; Peters, Stephen P; Alexis, Neil E; Woodruff, Prescott G; Han, MeiLan K; Meyers, Deborah A; Bleecker, Eugene R

2017-12-01

Increased concentrations of eosinophils in blood and sputum in chronic obstructive pulmonary disease (COPD) have been associated with increased frequency of exacerbations, reduced lung function, and corticosteroid responsiveness. We aimed to assess whether high eosinophil concentrations in either sputum or blood are associated with a severe COPD phenotype, including greater exacerbation frequency, and whether blood eosinophils are predictive of sputum eosinophils. We did a multicentre observational study analysing comprehensive baseline data from SPIROMICS in patients with COPD aged 40-80 years who had a smoking history of at least 20 pack-years, recruited from six clinical sites and additional subsites in the USA between Nov 12, 2010, and April 21, 2015. Inclusion criteria for this analysis were SPIROMICS baseline visit data with complete blood cell counts and, in a subset, acceptable sputum counts. We stratified patients on the basis of blood and sputum eosinophil concentrations and compared their demographic characteristics, as well as results from questionnaires, clinical assessments, and quantitative CT (QCT). We also analysed whether blood eosinophil concentrations reliably predicted sputum eosinophil concentrations. This study is registered with ClinicalTrials.gov (NCT01969344). Of the 2737 patients recruited to SPIROMICS, 2499 patients were smokers and had available blood counts, and so were stratified by mean blood eosinophil count: 1262 patients with low (<200 cells per μL) and 1237 with high (≥200 cells per μL) blood eosinophil counts. 827 patients were eligible for stratification by mean sputum eosinophil percentage: 656 with low (<1·25%) and 171 with high (≥1·25%) sputum eosinophil percentages. The high sputum eosinophil group had significantly lower median FEV 1 percentage predicted than the low sputum eosinophil group both before (65·7% [IQR 51·8-81·3] vs 75·7% [59·3-90·2], p<0·0001) and after (77·3% [63·1-88·5] vs 82·9% [67·8-95·9], p=0·001) bronchodilation. QCT density measures for emphysema and air trapping were significantly higher in the high sputum eosinophil group than the low sputum eosinophil group. Exacerbations requiring corticosteroids treatment were more common in the high versus low sputum eosinophil group (p=0·002). FEV 1 percentage predicted was significantly different between low and high blood eosinophil groups, but differences were less than those observed between the sputum groups. The high blood eosinophil group had slightly increased airway wall thickness (0·02 mm difference, p=0·032), higher St George Respiratory Questionnaire symptom scores (p=0·037), and increased wheezing (p=0·018), but no evidence of an association with COPD exacerbations (p=0·35) or the other indices of COPD severity, such as emphysema measured by CT density, COPD assessment test scores, Body-mass index, airflow Obstruction, Dyspnea, and Exercise index, or Global Initiative for Chronic Obstructive Lung Disease stage. Blood eosinophil counts showed a weak but significant association with sputum eosinophil counts (receiver operating characteristic area under the curve of 0·64, p<0·0001), but with a high false-discovery rate of 72%. In a large, well characterised cohort of former and current smoking patients with a broad range of COPD severity, high concentrations of sputum eosinophils were a better biomarker than high concentrations of blood eosinophils to identify a patient subgroup with more severe disease, more frequent exacerbations, and increased emphysema by QCT. Blood eosinophils alone were not a reliable biomarker for COPD severity or exacerbations, or for sputum eosinophils. Clinical trials targeting eosinophilic inflammation in COPD should consider assessing sputum eosinophils. National Institutes of Health, and National Heart, Lung, and Blood Institute. Copyright © 2017 Elsevier Ltd. All rights reserved.

Computerized analysis of cytology and fluorescence in situ hybridization (FISH) in induced sputum for lung cancer detection.

PubMed

Guber, Alexander; Greif, Joel; Rona, Roni; Fireman, Elizabeth; Madi, Lea; Kaplan, Tal; Yemini, Zipi; Gottfried, Maya; Katz, Ruth L; Daniely, Michal

2010-10-25

Lung cancer results from a multistep process, whereby genetic and epigenetic alterations lead to a malignant phenotype. Somatic mutations, deletions, and amplifications can be detected in the tumor itself, but they can also be found in histologically normal bronchial epithelium as a result of field cancerization. The present feasibility study describes a computer-assisted analysis of induced sputum employing morphology and fluorescence in situ hybridization (target-FISH), using 2 biomarkers located at chromosomes 3p22.1 and 10q22.3. Induced sputum samples were collected using a standardized protocol from 12 patients with lung cancer and from 15 healthy, nonsmoking controls. We used an automated scanning system that allows consecutive scans of morphology and FISH of the same slide. Cells derived for the lower airways were analyzed for the presence of genetic alterations in the 3p22.1 and 10q22.3 loci. The cutoff for a positive diagnosis was defined as >4% of cells showing genetic alterations. Eleven of 12 lung cancer patients and 12 of 15 controls were identified correctly, giving an overall sensitivity and specificity of 91.66% and 80%, respectively. This study describes a new technology for detecting lung cancer noninvasively in induced sputum via a combination of morphology and FISH analysis (target-FISH) using computer-assisted technology. This approach may potentially be utilized for mass screening of high-risk populations. © 2010 American Cancer Society.

Diagnostic value of sputum adenosine deaminase (ADA) level in pulmonary tuberculosis.

PubMed

Binesh, Fariba; Jalali, Hadi; Zare, Mohammad Reza; Behravan, Farhad; Tafti, Arefeh Dehghani; Behnaz, Fatemah; Tabatabaee, Mohammad; Shahcheraghi, Seyed Hossein

2016-06-01

Tuberculosis is still a considerable health problem in many countries. Rapid diagnosis of this disease is important, and adenosine deaminase (ADA) has been used as a diagnostic test. The aim of this study was to assess the diagnostic value of ADA in the sputum of patients with pulmonary tuberculosis. The current study included 40 patients with pulmonary tuberculosis (culture positive, smear ±) and 42 patients with non tuberculosis pulmonary diseases (culture negative). ADA was measured on all of the samples. The median value of ADA in non-tuberculosis patients was 2.94 (4.2) U/L and 4.01 (6.54) U/L in tuberculosis patients, but this difference was not statistically significant (p=0.100). The cut-off point of 3.1 U/L had a sensitivity of 61% and a specificity of 53%, the cut-off point of 2.81 U/L had a sensitivity of 64% and a specificity of 50% and the cut-off point of 2.78 U/L had a sensitivity of 65% and a specificity of 48%. The positive predictive values for cut-off points of 3.1, 2.81 and 2.78 U/L were 55.7%, 57.44% and 69.23%, respectively. The negative predictive values for the abovementioned cut-off points were 56.75%, 57.14% and 55.88%, respectively. Our results showed that sputum ADA test is neither specific nor sensitive. Because of its low sensitivity and specificity, determination of sputum ADA for the diagnosis of pulmonary tuberculosis is not recommended.

Effect of dairy farming system, herd, season, parity, and days in milk on modeling of the coagulation, curd firming, and syneresis of bovine milk.

PubMed

Bittante, G; Cipolat-Gotet, C; Malchiodi, F; Sturaro, E; Tagliapietra, F; Schiavon, S; Cecchinato, A

2015-04-01

The objectives of this study were to characterize the variation in curd firmness model parameters obtained from coagulating bovine milk samples, and to investigate the effects of the dairy system, season, individual farm, and factors related to individual cows (days in milk and parity). Individual milk samples (n = 1,264) were collected during the evening milking of 85 farms representing different environments and farming systems in the northeastern Italian Alps. The dairy herds were classified into 4 farming system categories: traditional system with tied animals (29 herds), modern dairy systems with traditional feeding based on hay and compound feed (30 herds), modern dairy system with total mixed ration (TMR) that included silage as a large proportion of the diet (9 herds), and modern dairy system with silage-free TMR (17 herds). Milk samples were analyzed for milk composition and coagulation properties, and parameters were modeled using curd firmness measures (CFt) collected every 15 s from a lacto-dynamographic analysis of 90 min. When compared with traditional milk coagulation properties (MCP), the curd firming measures showed greater variability and yielded a more accurate description of the milk coagulation process: the model converged for 93.1% of the milk samples, allowing estimation of 4 CFt parameters and 2 derived traits [maximum CF (CF(max)) and time from rennet addition to CF(max) (t(max))] for each sample. The milk samples whose CFt equations did not converge showed longer rennet coagulation times obtained from the model (RCT(eq)) and higher somatic cell score, and came from less-productive cows. Among the sources of variation tested for the CFt parameters, dairy herd system yielded the greatest differences for the contrast between the traditional farm and the 3 modern farms, with the latter showing earlier coagulation and greater instant syneresis rate constant (k(SR)). The use of TMR yielded a greater tmax because of a higher instant curd-firming rate constant (k(CF)). Season of sampling was found to be very important, yielding higher values during winter for all traits except k(CF) and k(SR). All CFt traits were affected by individual cow factors. For parity, milk produced by first-lactation cows showed higher k(CF) and k(SR), but delays in achieving CF(max). With respect to stage of lactation, RCT(eq) and potential asymptotic CF increased during the middle of lactation and stabilized thereafter, whereas the 2 instant rate constants presented the opposite pattern, with the lowest (k(CF)) and highest (k(SR)) values occurring in mid lactation. The new challenge offered by prolonging the test interval and individual modeling of milk technological properties allowed us to study the effects of parameters related to the environment and to individual cows. This novel strategy may be useful for investigating the genetic variability of these new coagulation traits. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Convergent Metabolic Specialization through Distinct Evolutionary Paths in Pseudomonas aeruginosa.

PubMed

La Rosa, Ruggero; Johansen, Helle Krogh; Molin, Søren

2018-04-10

Evolution by natural selection under complex and dynamic environmental conditions occurs through intricate and often counterintuitive trajectories affecting many genes and metabolic solutions. To study short- and long-term evolution of bacteria in vivo , we used the natural model system of cystic fibrosis (CF) infection. In this work, we investigated how and through which trajectories evolution of Pseudomonas aeruginosa occurs when migrating from the environment to the airways of CF patients, and specifically, we determined reduction of growth rate and metabolic specialization as signatures of adaptive evolution. We show that central metabolic pathways of three distinct Pseudomonas aeruginosa lineages coevolving within the same environment become restructured at the cost of versatility during long-term colonization. Cell physiology changes from naive to adapted phenotypes resulted in (i) alteration of growth potential that particularly converged to a slow-growth phenotype, (ii) alteration of nutritional requirements due to auxotrophy, (iii) tailored preference for carbon source assimilation from CF sputum, (iv) reduced arginine and pyruvate fermentation processes, and (v) increased oxygen requirements. Interestingly, although convergence was evidenced at the phenotypic level of metabolic specialization, comparative genomics disclosed diverse mutational patterns underlying the different evolutionary trajectories. Therefore, distinct combinations of genetic and regulatory changes converge to common metabolic adaptive trajectories leading to within-host metabolic specialization. This study gives new insight into bacterial metabolic evolution during long-term colonization of a new environmental niche. IMPORTANCE Only a few examples of real-time evolutionary investigations in environments outside the laboratory are described in the scientific literature. Remembering that biological evolution, as it has progressed in nature, has not taken place in test tubes, it is not surprising that conclusions from our investigations of bacterial evolution in the CF model system are different from what has been concluded from laboratory experiments. The analysis presented here of the metabolic and regulatory driving forces leading to successful adaptation to a new environment provides an important insight into the role of metabolism and its regulatory mechanisms for successful adaptation of microorganisms in dynamic and complex environments. Understanding the trajectories of adaptation, as well as the mechanisms behind slow growth and rewiring of regulatory and metabolic networks, is a key element to understand the adaptive robustness and evolvability of bacteria in the process of increasing their in vivo fitness when conquering new territories. Copyright © 2018 La Rosa et al.

THE IMPACT OF LATE-LIFE ANXIETY AND DEPRESSION ON COGNITIVE FLEXIBILITY AND COGNITIVE RESTRUCTURING SKILL ACQUISITION.

PubMed

Johnco, Carly; Wuthrich, Viviana M; Rapee, Ronald M

2015-10-01

Studies examining treatment moderators and mediators in late-life anxiety and depression are sparse. Executive functioning skills decrease with age, and are poorer in the context of anxiety and depression. One of the key cognitive behavioral therapy techniques for anxiety and depression is cognitive restructuring (CR), which teaches people to identify and dispute maladaptive thoughts. There is evidence that cognitive flexibility (CF), one aspect of executive functioning, has a negative impact on CR skill acquisition in nonclinical older adults, and this warrants extension in a clinical sample. This study assessed CR skill acquisition in a clinical sample of 47 older adults with anxiety and depression and 53 nonclinical controls during an experimental paradigm, and investigated the influence of CF on this relationship. A battery of neuropsychological tests assessing CF were administered and CR was learned during a brief intervention. The clinical sample showed poorer CF on some measures, as well as poorer CR quality and efficacy (reduction in subjective distress). CF partially mediated the relationship between clinical status and CR quality, and between clinical status and CR efficacy. These results provide preliminary evidence that older adults with anxiety and depression are worse at learning and benefiting from CR with a brief intervention and this is partially due to having poorer CF skills. These findings warrant further examination in a treatment context to assess whether CR skill acquisition improves over treatment. © 2015 Wiley Periodicals, Inc.

Benchmark Evaluation of True Single Molecular Sequencing to Determine Cystic Fibrosis Airway Microbiome Diversity.

PubMed

Hahn, Andrea; Bendall, Matthew L; Gibson, Keylie M; Chaney, Hollis; Sami, Iman; Perez, Geovanny F; Koumbourlis, Anastassios C; McCaffrey, Timothy A; Freishtat, Robert J; Crandall, Keith A

2018-01-01

Cystic fibrosis (CF) is an autosomal recessive disease associated with recurrent lung infections that can lead to morbidity and mortality. The impact of antibiotics for treatment of acute pulmonary exacerbations on the CF airway microbiome remains unclear with prior studies giving conflicting results and being limited by their use of 16S ribosomal RNA sequencing. Our primary objective was to validate the use of true single molecular sequencing (tSMS) and PathoScope in the analysis of the CF airway microbiome. Three control samples were created with differing amounts of Burkholderia cepacia , Pseudomonas aeruginosa , and Prevotella melaninogenica , three common bacteria found in cystic fibrosis lungs. Paired sputa were also obtained from three study participants with CF before and >6 days after initiation of antibiotics. Antibiotic resistant B. cepacia and P. aeruginosa were identified in concurrently obtained respiratory cultures. Direct sequencing was performed using tSMS, and filtered reads were aligned to reference genomes from NCBI using PathoScope and Kraken and unique clade-specific marker genes using MetaPhlAn. A total of 180-518 K of 6-12 million filtered reads were aligned for each sample. Detection of known pathogens in control samples was most successful using PathoScope. In the CF sputa, alpha diversity measures varied based on the alignment method used, but similar trends were found between pre- and post-antibiotic samples. PathoScope outperformed Kraken and MetaPhlAn in our validation study of artificial bacterial community controls and also has advantages over Kraken and MetaPhlAn of being able to determine bacterial strains and the presence of fungal organisms. PathoScope can be confidently used when evaluating metagenomic data to determine CF airway microbiome diversity.

Rapid detection of mecA and spa by the loop-mediated isothermal amplification (LAMP) method.

PubMed

Koide, Y; Maeda, H; Yamabe, K; Naruishi, K; Yamamoto, T; Kokeguchi, S; Takashiba, S

2010-04-01

To develop a detection assay for staphylococcal mecA and spa by using loop-mediated isothermal amplification (LAMP) method. Staphylococcus aureus and other related species were subjected to the detection of mecA and spa by both PCR and LAMP methods. The LAMP successfully amplified the genes under isothermal conditions at 64 degrees C within 60 min, and demonstrated identical results with the conventional PCR methods. The detection limits of the LAMP for mecA and spa, by gel electrophoresis, were 10(2) and 10 cells per tube, respectively. The naked-eye inspections were possible with 10(3) and 10 cells for detection of mecA and spa, respectively. The LAMP method was then applied to sputum and dental plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples, although frequency in detection of mecA and spa by the LAMP was relatively lower for the sputum samples when compared to the PCR methods. Application of the LAMP enabled a rapid detection assay for mecA and spa. The assay may be applicable to clinical plaque samples. The LAMP offers an alternative detection assay for mecA and spa with a great advantage of the rapidity.

RNA sequencing data from neutrophils of patients with cystic fibrosis reveals potential for developing biomarkers for pulmonary exacerbations.

PubMed

Jiang, Kaiyu; Poppenberg, Kerry E; Wong, Laiping; Chen, Yanmin; Borowitz, Drucy; Goetz, Danielle; Sheehan, Daniel; Frederick, Carla; Tutino, Vincent M; Meng, Hui; Jarvis, James N

2018-06-22

There is no effective way to predict cystic fibrosis (CF) pulmonary exacerbations (CFPE) before they become symptomatic or to assess satisfactory treatment responses. RNA sequencing of peripheral blood neutrophils from CF patients before and after therapy for CFPE was used to create transcriptome profiles. Transcripts with an average transcripts per million (TPM) level > 1.0 and a false discovery rate (FDR) < 0.05 were used in a cosine K-nearest neighbor (KNN) model. Real time PCR was used to corroborate RNA sequencing expression differences in both neutrophils and whole blood samples from an independent cohort of CF patients. Furthermore, sandwich ELISA was conducted to assess plasma levels of MRP8/14 complexes in CF patients before and after therapy. We found differential expression of 136 transcripts and 83 isoforms when we compared neutrophils from CF patients before and after therapy (>1.5 fold change, FDR-adjusted P < 0.05). The model was able to successfully separate CF flare samples from those taken from the same patients in convalescence with an accuracy of 0.75 in both the training and testing cohorts. Six differently expressed genes were confirmed by real time PCR using both isolated neutrophils and whole blood from an independent cohort of CF patients before and after therapy, even though levels of myeloid related protein MRP8/14 dimers in plasma of CF patients were essentially unchanged by therapy. Our findings demonstrate the potential of machine learning approaches for classifying disease states and thus developing sensitive biomarkers that can be used to monitor pulmonary disease activity in CF. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Void statistics of the CfA redshift survey

NASA Technical Reports Server (NTRS)

Vogeley, Michael S.; Geller, Margaret J.; Huchra, John P.

1991-01-01

Clustering properties of two samples from the CfA redshift survey, each containing about 2500 galaxies, are studied. A comparison of the velocity distributions via a K-S test reveals structure on scales comparable with the extent of the survey. The void probability function (VPF) is employed for these samples to examine the structure and to test for scaling relations in the galaxy distribution. The galaxy correlation function is calculated via moments of galaxy counts. The shape and amplitude of the correlation function roughly agree with previous determinations. The VPFs for distance-limited samples of the CfA survey do not match the scaling relation predicted by the hierarchical clustering models. On scales not greater than 10/h Mpc, the VPFs for these samples roughly follow the hierarchical pattern. A variant of the VPF which uses nearly all the data in magnitude-limited samples is introduced; it accounts for the variation of the sampling density with velocity in a magnitude-limited survey.

Void statistics of the CfA redshift survey

NASA Astrophysics Data System (ADS)

Vogeley, Michael S.; Geller, Margaret J.; Huchra, John P.

1991-11-01

Clustering properties of two samples from the CfA redshift survey, each containing about 2500 galaxies, are studied. A comparison of the velocity distributions via a K-S test reveals structure on scales comparable with the extent of the survey. The void probability function (VPF) is employed for these samples to examine the structure and to test for scaling relations in the galaxy distribution. The galaxy correlation function is calculated via moments of galaxy counts. The shape and amplitude of the correlation function roughly agree with previous determinations. The VPFs for distance-limited samples of the CfA survey do not match the scaling relation predicted by the hierarchical clustering models. On scales not greater than 10/h Mpc, the VPFs for these samples roughly follow the hierarchical pattern. A variant of the VPF which uses nearly all the data in magnitude-limited samples is introduced; it accounts for the variation of the sampling density with velocity in a magnitude-limited survey.

Effects of doxycycline on local and systemic inflammation in stable COPD patients, a randomized clinical trial.

PubMed

Prins, Hendrik J; Daniels, Johannes M A; Lindeman, Jan H; Lutter, René; Boersma, Wim G

2016-01-01

Neutrophilic inflammation plays a causal role in Chronic Obstructive Pulmonary Disease (COPD). Neutrophil derived myeloperoxidase(MPO) matrix metalloproteinases(MMP's), and elastases are thought to contribute to the perpetuation of the disease. The tetracycline analogue doxycycline has been shown to inhibit neutrophil-mediated inflammation. It was thus reasoned that doxycycline may attenuate neutrophil-mediated inflammation in COPD. In this double blind randomized controlled trial the effect of a 3-week course of doxycycline on sputum and systemic inflammatory parameters was evaluated in stable COPD patients. In order to exclude inflammation by bacterial colonisation patients must have 2 negative sputum cultures in the previous year. The effect of doxycycline treatment on inflammatory markers (TNF-α, IL-1β and IL-6) and neutrophil specific markers in sputum (MPO, MMP's, and IL-8) and serum C-reactive protein was evaluated. Sputum was obtained by sputum induction with hypertonic saline. A total of 41 patients were included. Ten patients were excluded as they were not able to produce sputum at the first or second visit. Baseline characteristics were similar in the two groups. In the remaining patients doxycycline did not influence sputum MPO concentrations. Also MMP-8 and 9, IL-6 and IL-8 concentrations as well as lung function parameters were not affected by doxycycline. Systemic inflammation by means of CRP was also not influenced by doxycycline. A three week course of doxycycline did not influence MPO sputum levels nor any of the other inflammatory sputum and systemic markers. ClinicalTrials.gov; No.: NCT00857038 URL: clinicaltrials.gov. Copyright © 2015 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saccomanno, G.

This work supported by the United States of Energy, continues to add data on the health affects of cigarette smoking and radon exposure on uranium miners. Since the last Technical Progress Report in July or 1991, 537 sputum cytology samples have been collected on the 300 uranium workers in the surveillance study. To date there are 436 lung cancer cases in the Uranium Miner Tumor Registry with diagnostic slides from surgery and/or autopsy; an additional 40 cases have been diagnosed with sputum cytology only. In March of 1991 the Geno Saccomanno Uranium Workers Archive was established at St. Mary`s Hospitalmore » and Medical Center as a depository for biological specimens and epidemiological data from the 17,700 uranium miners who have been a part or the study.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saccomanno, G.

This work supported by the United States of Energy, continues to add data on the health affects of cigarette smoking and radon exposure on uranium miners. Since the last Technical Progress Report in July or 1991, 537 sputum cytology samples have been collected on the 300 uranium workers in the surveillance study. To date there are 436 lung cancer cases in the Uranium Miner Tumor Registry with diagnostic slides from surgery and/or autopsy; an additional 40 cases have been diagnosed with sputum cytology only. In March of 1991 the Geno Saccomanno Uranium Workers Archive was established at St. Mary's Hospitalmore » and Medical Center as a depository for biological specimens and epidemiological data from the 17,700 uranium miners who have been a part or the study.« less

[Lung cancer in survivors of radiation exposure at the Chernobyl nuclear disaster].

PubMed

Zubovskiĭ, G A; Khrisanfov, S A

2003-01-01

Epidemiology, diagnostics and therapy of lung cancer in the aftermath of Chernobyl nuclear disaster are discussed on the basis of the data released by the Federal Expert Committee. Lung cancer appeared to be the main death-causing factor. The disease was far advanced (stage IIIb and IV) in 85% of cases. The effectiveness of diagnosis can be raised if such compulsory measures as annual bronchoscopic screenings and sputum counts are carried out. Atypical cell counts have to be run in the sputum samples from all the survivors who took part in the salvaging operations of 1986-1987 and are suffering acute and chronic respiratory diseases. Particular attention should be paid to those who were working under severe dust-pollution conditions in summer.

Genetic variation and effects of candidate-gene polymorphisms on coagulation properties, curd firmness modeling and acidity in milk from Brown Swiss cows.

PubMed

Cecchinato, A; Chessa, S; Ribeca, C; Cipolat-Gotet, C; Bobbo, T; Casellas, J; Bittante, G

2015-07-01

The aims of this study were to estimate the genetic variation of traditional milk coagulation properties (MCPs), milk acidity, curd firmness (CF) modeled on time t (CF(t) ; comprising: RCT(eq), rennet coagulation time estimated from the equation; CF(P), the asymptotic potential curd firmness; k(CF), the curd firming instant rate constant; and k(SR), the syneresis instant rate constant) and maximum CF traits (MCF; comprising CF(max), the maximum CF value; and tmax, the time of attainment). Furthermore, we investigated 96 single nucleotide polymorphisms (SNPs) from 54 candidate genes, testing their associations with the above-listed traits. Milk and blood samples were collected from 1271 cows (each sampled once) from 85 herds. Genotyping was performed using a custom Illumina VeraCode GoldenGate approach. A Bayesian linear animal model (including the effects of herd, days in milk, parity and additive polygenic effects) was used to estimate the genetic parameters of the studied traits. The same model with the addition of the SNP genotype effect was used for our association analysis. The heritability estimates of CF t and the MCF traits (RCT(eq)=0.258; k(CF)=0.230; CF(max)=0.191; t(max)=0.278) were similar to those obtained using traditional MCPs (0.187 to 0.267), except for the lower estimates for CF(P) (0.064) and k(SR) (0.077). A total of 13 of the 51 tested SNPs had relevant additive effects on at least one trait. We observed associations between MCPs and SNPs in the genes encoding ATP-binding cassette sub-family G member 2 (ABCG2), chemokine ligand 2 (CCL2), growth hormone 1 (GH1), prolactin (PRL) and toll-like receptor 2 (TLR2). Whereas, CF(t) and the MCF traits were associated with polymorphisms in the α-s1-casein (CSN1S1), β-casein (CSN2), GH1, oxidized low-density lipoprotein receptor 1 (OLR1), phospholipase C β1 (PLCB1), PRL and signal transducer and activator of transcription 5A (STAT5A) genes.

OMNIgene.SPUTUM suppresses contaminants while maintaining Mycobacterium tuberculosis viability and obviates cold-chain transport.

PubMed

Azam, Khalide; Cadir, Nureisha; Madeira, Carla; Gillespie, Stephen H; Sabiiti, Wilber

2018-01-01

Tuberculosis (TB) diagnostics are centralised, requiring long-distance transportation of specimens in most resource-limited settings. We evaluated the ability of OMNIgene.SPUTUM (OM-S) to obviate cold-chain transport of TB specimens. A two-arm (same-day and after 5 days sample processing) study was conducted to assess contamination rates and Mycobacterium tuberculosis viability in OM-S-treated samples against the standard decontamination procedure (SDP) in Mozambique, using Lowenstein Jensen (LJ) and mycobacterial growth indicator tube (MGIT) culture and molecular bacterial load assay. 270 specimens were processed using OM-S and SDP in same-day and 5-day arms. Contamination was lower in OM-S-treated than SDP-treated cultures: 12% versus 15% and 2% versus 27% in the same-day and 5-day arms, respectively. M. tuberculosis recovery in OM-S-treated LJ cultures was 10% and 56% higher in the same-day and 5-day arms, respectively, than SDP-treated cultures, but lower in MGIT (52% and 28% lower in the same-day and 5-day arms, respectively). M. tuberculosis viable count was 1log estimated CFU·mL -1 lower in 5-day OM-S-treated sputa. OM-S was more effective at liquefying sputum with a shorter sample processing time: 22 min for culture. OM-S is simple to use and has demonstrated a high potency to suppress contaminants, maintenance of viability at ambient temperatures and higher M. tuberculosis recovery, particularly in the solid LJ cultures. Optimisation of OM-S to achieve higher MGIT culture positivity and shorter time to result will increase its application and utility in the clinical management of TB.

OMNIgene.SPUTUM suppresses contaminants while maintaining Mycobacterium tuberculosis viability and obviates cold-chain transport

PubMed Central

Azam, Khalide; Cadir, Nureisha; Madeira, Carla; Gillespie, Stephen H.; Sabiiti, Wilber

2018-01-01

Tuberculosis (TB) diagnostics are centralised, requiring long-distance transportation of specimens in most resource-limited settings. We evaluated the ability of OMNIgene.SPUTUM (OM-S) to obviate cold-chain transport of TB specimens. A two-arm (same-day and after 5 days sample processing) study was conducted to assess contamination rates and Mycobacterium tuberculosis viability in OM-S-treated samples against the standard decontamination procedure (SDP) in Mozambique, using Lowenstein Jensen (LJ) and mycobacterial growth indicator tube (MGIT) culture and molecular bacterial load assay. 270 specimens were processed using OM-S and SDP in same-day and 5-day arms. Contamination was lower in OM-S-treated than SDP-treated cultures: 12% versus 15% and 2% versus 27% in the same-day and 5-day arms, respectively. M. tuberculosis recovery in OM-S-treated LJ cultures was 10% and 56% higher in the same-day and 5-day arms, respectively, than SDP-treated cultures, but lower in MGIT (52% and 28% lower in the same-day and 5-day arms, respectively). M. tuberculosis viable count was 1log estimated CFU·mL−1 lower in 5-day OM-S-treated sputa. OM-S was more effective at liquefying sputum with a shorter sample processing time: 22 min for culture. OM-S is simple to use and has demonstrated a high potency to suppress contaminants, maintenance of viability at ambient temperatures and higher M. tuberculosis recovery, particularly in the solid LJ cultures. Optimisation of OM-S to achieve higher MGIT culture positivity and shorter time to result will increase its application and utility in the clinical management of TB. PMID:29479537

Detection of Mycobacterium tuberculosis based on H37R(v) binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis.

PubMed

Yang, Hua; Qin, Lianhua; Wang, Yilong; Zhang, Bingbo; Liu, Zhonghua; Ma, Hui; Lu, Junmei; Huang, Xiaochen; Shi, Donglu; Hu, Zhongyi

2015-01-01

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10(3) colony forming units (CFU)/mL H37R(v) was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10(3) CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.

Detection of Mycobacterium tuberculosis based on H37Rv binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis

PubMed Central

Yang, Hua; Qin, Lianhua; Wang, Yilong; Zhang, Bingbo; Liu, Zhonghua; Ma, Hui; Lu, Junmei; Huang, Xiaochen; Shi, Donglu; Hu, Zhongyi

2015-01-01

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl–Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 103 colony forming units (CFU)/mL H37Rv was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 103 CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples. PMID:25565805

[Sputum viscosity and pulmonary function measurements during a one-week parenteral treatment with a standardized oxidation product of oil of turpentine and terpin hydrate].

PubMed

Löllgen-Horres, I; Löllgen, H

1976-01-01

In 23 patients with chronic obstructive lung diseases, viscosity, airway resistance, arterial blood gases and acid-base balance, and sputum aspect were measured before and after one-week treatment with Ozothin, a substance from oxidation products of ol. terebinth. and terpinum hydratum. Within this time, viscosity of the sputum was reduced, airway resistance decreased, and arterial oxygen pressure slightly increased, whereas arterial carbon dioxide tension obvious change of sputum aspect could be observed. Correlation calculations revealed no significant relations between viscosity and the above cited lung function values. The results indicate that administration of Ozothin may liquefy viscous secretion and reduce sputum viscosity.

Monitoring of toxic elements present in sludge of industrial waste using CF-LIBS.

PubMed

Kumar, Rohit; Rai, Awadhesh K; Alamelu, Devanathan; Aggarwal, Suresh K

2013-01-01

Industrial waste is one of the main causes of environmental pollution. Laser-induced breakdown spectroscopy (LIBS) was applied to detect the toxic metals in the sludge of industrial waste water. Sludge on filter paper was obtained after filtering the collected waste water samples from different sections of a water treatment plant situated in an industrial area of Kanpur City. The LIBS spectra of the sludge samples were recorded in the spectral range of 200 to 500 nm by focusing the laser light on sludge. Calibration-free laser-induced breakdown spectroscopy (CF-LIBS) technique was used for the quantitative measurement of toxic elements such as Cr and Pb present in the sample. We also used the traditional calibration curve approach to quantify these elements. The results obtained from CF-LIBS are in good agreement with the results from the calibration curve approach. Thus, our results demonstrate that CF-LIBS is an appropriate technique for quantitative analysis where reference/standard samples are not available to make the calibration curve. The results of the present experiment are alarming to the people living nearby areas of industrial activities, as the concentrations of toxic elements are quite high compared to the admissible limits of these substances.

Real-Life Clinical Practice of Using the Xpert MTB/RIF Assay in Thailand.

PubMed

Kawkitinarong, Kamon; Suwanpimolkul, Gompol; Kateruttanakul, Pairaj; Manosuthi, Weerawat; Ubolyam, Sasiwimol; Sophonphan, Jiratchaya; Avihingsanon, Anchalee; Ruxrungtham, Kiat

2017-05-15

Delayed diagnosis of tuberculosis (TB) and drug-resistant TB are major challenges of TB control in Thailand. This study assessed the practicality of the Xpert MTB/RIF assay in a real-life setting with high prevalence of human immunodeficiency virus (HIV) infection and pulmonary tuberculosis (PTB). This prospective study was conducted at 3 large tertiary care hospitals. Patients who had suspected PTB were enrolled into the study. Expectorated sputum samples were sent for staining, mycobacterial culture, and Xpert MTB/RIF. Four hundred ninety-four patients were enrolled. From 355 cases with final diagnosis of PTB, 263 (71.8%) had definite diagnosis and 92 cases had probable diagnosis. Among TB culture-positive cases, Xpert MTB/RIF had 100% and 81% sensitivity in sputum smear-positive and smear-negative groups, respectively. The specificity was 95.7%. The sensitivity and positive predictive value of Xpert MTB/RIF in culture-negative but clinically diagnosed PTB was 37.8% and 83.8%, respectively. Centrifugation was required in 59% cases with scanty sputum. Five cases were false-positive by Xpert MTB/RIF in patients with nontuberculous mycobacteria, old PTB scar, and immune reconstitution syndrome. Discordant rifampicin susceptibility results of Xpert MTB/RIF and mycobacteria growth indicator tube (MGIT) were confirmed by using rpoB gene sequencing, which raised the sensitivity of Xpert MTB/RIF in detecting rifampicin resistance to 93.8%. Xpert MTB/RIF is an effective tool in diagnosing PTB but will be more cost-effective for sputum-negative patients and in settings with high prevalence of rifampicin resistance. Early diagnosis of TB results in early treatment and implementation of strategies to limit spreading of TB. Sputum centrifugation may increase the yield of Xpert MTB/RIF. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Usefulness of induced sputum eosinophil count to assess severity and treatment outcome in asthma patients

PubMed Central

Bandyopadhyay, Ankan; Roy, Partha P.; Saha, Kaushik; Chakraborty, Semanti; Jash, Debraj; Saha, Debabrata

2013-01-01

Context: Currently treatment decisions in asthma are governed by clinical assessment and spirometry. Sputum eosinophil, being a marker of airway inflammation, can serve as a tool for assessing severity and response to treatment in asthma patients. Aims: To establish correlation between change in sputum eosinophil count and forced expiratory volume in one second (FEV1)% predicted value of asthma patients in response to treatment. In this study, we also predicted prognosis and treatment outcome of asthma patients from baseline sputum eosinophil count. Settings and Design: A longitudinal study was conducted to determine the treatment outcome among newly diagnosed asthma patients who were classified into A (n = 80) and B (n = 80) groups on the basis of initial sputum eosinophil count (A ≥ 3% and B < 3%). Materials and Methods: After starting treatment according to Global Initiative for Asthma Guideline, both A and B groups were evaluated every 15 days interval for the 1st month and monthly thereafter for a total duration of 12 months. In each follow-up visit detailed history, induced sputum eosinophil count and spirometry were done to evaluate severity and treatment outcome. Results: FEV1% predicted of group A asthma patients gradually increased and sputum eosinophil count gradually decreased on treatment. Longer time was required to achieve satisfactory improvement (FEV1% predicted) in asthma patients with sputum eosinophil count ≥3%. There was statistically significant negative correlation between FEV1% predicted and sputum eosinophil count (%) in of group A patients in each follow-up visit, with most significant negative correlation found in 8th visit (r = −0.9237 and P = < 0.001). Change in mean FEV1% (predicted) from baseline showed strong positive correlation (r = 0.976) with change in reduction of mean sputum eosinophil count at each follow-up visits in group A patients. Conclusions: Sputum eosinophil count, being an excellent biomarker of airway inflammation, can serve as a useful marker to assess disease severity, treatment outcome, and prognosis in asthma patients. PMID:23741092

Magnetism in californium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moore, J.R.

1988-03-01

A SQUID-based magnetic susceptometer has been constructed for studying small radioactive samples at temperatures below 350 K and in magnetic fields up to 50 kilogauss. The device has been used to study californium (element 98) in a number of solid-state forms: the dhcp metal, several oxides (Cf/sub 2/O/sub 3/ in both the bcc and monoclinic structures, Cf/sub 7/O/sub 12/, CfO/sub 2/ and BaCfO/sub 3/), several monopnictides (CfN, CfAs and CfSb) and the trichloride (in both the hexagonal and orthorhombic structures). All of these materials were studied in polycrystalline form, and hexagonal CfCl/sub 3/ was studied in single-crystal form as well.more » The susceptometer has the sensitivity to measure samples containing less than 10 micrograms of californium. The magnetic susceptibilities of all of the californium materials at temperatures above about 100 K are described well by the Curie-Weiss relationship. This behavior is consistent with the assumption that the magnetic 5f electrons are localized and that the paramagnetic behavior can be interpreted in terms of the properties of the free ion. The measured values of the effective paramagnetic moment, ..mu../sub eff/, for all the californium materials that were studied are reasonably consistent with theoretical values based on intermediate coupling models. All of the californium materials showed some indications of cooperative magnetic effects. The dhcp metal was observed to order ferromagnetically at 52 K, and all of the californium compounds studied showed signs of antiferromagnetic ordering, mostly at temperatures below 25 K. 91 refs., 50 figs., 19 tabs.« less

Burnout, compassion fatigue, compassion satisfaction, and secondary traumatic stress in trauma nurses.

PubMed

Hinderer, Katherine A; VonRueden, Kathryn T; Friedmann, Erika; McQuillan, Karen A; Gilmore, Rebecca; Kramer, Betsy; Murray, Mary

2014-01-01

The relationship of burnout (BO), compassion fatigue (CF), compassion satisfaction (CS), and secondary traumatic stress (STS) to personal/environmental characteristics, coping mechanisms, and exposure to traumatic events was explored in 128 trauma nurses. Of this sample, 35.9% had scores consistent with BO, 27.3% reported CF, 7% reported STS, and 78.9% had high CS scores. High BO and high CF scores predicted STS. Common characteristics correlating with BO, CF, and STS were negative coworker relationships, use of medicinals, and higher number of hours worked per shift. High CS correlated with greater strength of supports, higher participation in exercise, use of meditation, and positive coworker relationships. Caring for trauma patients may lead to BO, CF, and STS; identifying predictors of these can inform the development of interventions to mitigate or minimize BO, CF, and STS in trauma nurses.

Enhancement of ionization efficiency of mass spectrometric analysis from non-electrospray ionization friendly solvents with conventional and novel ionization techniques.

PubMed

Jiang, Ping; Lucy, Charles A

2015-10-15

Electrospray ionization mass spectrometry (ESI-MS) has significantly impacted the analysis of complex biological and petroleum samples. However ESI-MS has limited ionization efficiency for samples in low dielectric and low polarity solvents. Addition of a make-up solvent through a T union or electrospray solvent through continuous flow extractive desorption electrospray ionization (CF-EDESI) enable ionization of analytes in non-ESI friendly solvents. A conventional make-up solvent addition setup was used and a CF-EDESI source was built for ionization of nitrogen-containing standards in hexane or hexane/isopropanol. Factors affecting the performance of both sources have been investigated and optimized. Both the make-up solvent addition and CF-EDESI improve the ionization efficiency for heteroatom compounds in non-ESI friendly solvents. Make-up solvent addition provides higher ionization efficiency than CF-EDESI. Neither the make-up solvent addition nor the CF-EDESI eliminates ionization suppression of nitrogen-containing compounds caused by compounds of the same chemical class. Copyright © 2015 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Archer, V.E.; Fullmer, C.D.; Castle, C.H.

Although acute effects from exposure to SO/sub 2/ have frequently been observed at low levels in acute exposure experimental studies, it was not known whether or not such effects occur among workers chronically exposed at ranges of 0.3 to 4 ppM of SO/sub 2/. Measurements of FVC, FEV/sub 1/, FEF/sub 50/, FEF/sub 75/ and FEF/sub 50-75/, and closing volume were made before and after the workshift for copper smelter workers and controls. Sputum samples for cytological examination were collected. Mean FEV/sub 1/ and FVC were significantly decreased during a day's work in the smelter. Significantly more smelter workers had amore » decrease in FEV/sub 1/ and FEF/sub 50/ during the day than did controls. More of the smelter workers felt chest tightness. No change in closing volumes was seen. Smelter workers tended to have a higher percentage of sputum samples with moderate and marked atypia than did controls but the difference was not statistically significant.« less

Associations between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits in dairy cows.

PubMed

Bobbo, T; Ruegg, P L; Stocco, G; Fiore, E; Gianesella, M; Morgante, M; Pasotto, D; Bittante, G; Cecchinato, A

2017-06-01

The aim of this study was to investigate associations between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits. Forty-one multibreed herds were selected for the study, and composite milk samples were collected from 1,508 cows belonging to 3 specialized dairy breeds (Holstein Friesian, Brown Swiss, and Jersey) and 3 dual-purpose breeds of Alpine origin (Simmental, Rendena, and Grey Alpine). Milk composition [i.e., fat, protein, casein, lactose, pH, urea, and somatic cell count (SCC)] was analyzed, and separation of protein fractions was performed by reversed-phase high performance liquid chromatography. Eleven coagulation traits were measured: 5 traditional milk coagulation properties [time from rennet addition to milk gelation (RCT, min), curd-firming rate as the time to a curd firmness (CF) of 20 mm (k 20 , min), and CF at 30, 45, and 60 min from rennet addition (a 30 , a 45 , and a 60 , mm)], and 6 new curd firming and syneresis traits [potential asymptotical CF at an infinite time (CF P , mm), curd-firming instant rate constant (k CF , % × min -1 ), curd syneresis instant rate constant (k SR , % × min -1 ), modeled RCT (RCT eq , min), maximum CF value (CF max, mm), and time at CF max (t max , min)]. We also measured 3 cheese yield traits, expressing the weights of total fresh curd (%CY CURD ), dry matter (%CY SOLIDS ), and water (%CY WATER ) in the curd as percentages of the weight of the processed milk, and 4 nutrient recovery traits (REC PROTEIN , REC FAT , REC SOLIDS , and REC ENERGY ), representing the percentage ratio between each nutrient in the curd and milk. Milk samples with SCC > 100,000 cells/mL were subjected to bacteriological examination. All samples were divided into 7 clusters of udder health (UH) status: healthy (cows with milk SCC < 100,000 cells/mL and uncultured); culture-negative samples with low, medium, or high SCC; and culture-positive samples divided into contagious, environmental, and opportunistic intramammary infection (IMI). Data were analyzed using a linear mixed model. Significant variations in the casein to protein ratio and lactose content were observed in all culture-positive samples and in culture-negative samples with medium to high SCC compared to normal milk. No differences were observed among contagious, environmental, and opportunistic pathogens, suggesting an effect of inflammation rather than infection. The greatest impairment in milk quantity and composition, clotting ability, and cheese production was observed in the 2 UH status groups with the highest milk SCC (i.e., contagious IMI and culture-negative samples with high SCC), revealing a discrepancy between the bacteriological results and inflammatory status, and thus confirming the importance of SCC as an indicator of udder health and milk quality. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Surface modification of epoxy resin using He/CF4 atmospheric pressure plasma jet for flashover withstanding characteristics improvement in vacuum

NASA Astrophysics Data System (ADS)

Chen, Sile; Wang, Shuai; Wang, Yibo; Guo, Baohong; Li, Guoqiang; Chang, Zhengshi; Zhang, Guan-Jun

2017-08-01

For enhancing the surface electric withstanding strength of insulating materials, epoxy resin (EP) samples are treated by atmospheric pressure plasma jet (APPJ) with different time interval from 0 to 300s. Helium (He) and tetrafluoromethane (CF4) mixtures are used as working gases with the concentration of CF4 ranging 0%-5%, and when CF4 is ∼3%, the APPJ exhibits an optimal steady state. The flashover withstanding characteristics of modified EP in vacuum are greatly improved under appropriate APPJ treatment conditions. The surface properties of EP samples are evaluated by surface roughness, scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS) and water contact angle. It is considered that both physical and chemical effects lead to the enhancement of flashover strength. The physical effect is reflected in the increase of surface roughness, while the chemical effect is reflected in the graft of fluorine groups.

Effects of an Antioxidant-enriched Multivitamin in Cystic Fibrosis: Randomized, Controlled, Multicenter Trial.

PubMed

Sagel, Scott D; Khan, Umer; Jain, Raksha; Graff, Gavin; Daines, Cori L; Dunitz, Jordan M; Borowitz, Drucy; Orenstein, David M; Abdulhamid, Ibrahim; Noe, Julie; Clancy, John P; Slovis, Bonnie; Rock, Michael J; McCoy, Karen S; Strausbaugh, Steven; Livingston, Floyd R; Papas, Konstantinos A; Shaffer, Michele L

2018-04-24

Cystic fibrosis (CF) is characterized by dietary antioxidant deficiencies, which may contribute to an oxidant-antioxidant imbalance and oxidative stress. Evaluate the effects of an oral antioxidant-enriched multivitamin supplement on antioxidant concentrations, markers of inflammation and oxidative stress, and clinical outcomes. In this investigator-initiated, multicenter, randomized, double-blind, controlled trial, 73 pancreatic insufficient CF subjects 10 years of age and older with an FEV1 between 40-100% predicted were randomized to 16 weeks of an antioxidant-enriched multivitamin or control multivitamin without antioxidant enrichment. Endpoints included systemic antioxidant concentrations, markers of inflammation and oxidative stress, clinical outcomes (pulmonary exacerbations, anthropometric measures, pulmonary function), safety and tolerability. Change in sputum myeloperoxidase concentration over 16 weeks, the primary efficacy endpoint, was not significantly different between the treated and control groups. Systemic antioxidant concentrations (β-carotene, CoQ10, γ-tocopherol, lutein) significantly increased in the antioxidant treated group (p<0.001 for each), while circulating calprotectin and myeloperoxidase decreased in the treated group compared to the control group at week 4. The treated group had a lower risk of first pulmonary exacerbation requiring antibiotics than the control group (adjusted hazard ratio=0.50, p=0.04). Lung function and growth endpoints did not differ between groups. Adverse events and tolerability were similar between groups. Antioxidant supplementation was safe and well tolerated, resulting in increased systemic antioxidant concentrations and modest reductions in systemic inflammation after 4 weeks. Antioxidant treatment was also associated with a lower risk of first pulmonary exacerbation. Clinical trial registration available at www.clinicaltrials.gov, ID NCT01859390.

Anti-citrullinated protein antibodies are associated with neutrophil extracellular traps in the sputum in relatives of rheumatoid arthritis patients

PubMed Central

Demoruelle, M. Kristen; Harrall, Kylie K.; Ho, Linh; Purmalek, Monica M.; Seto, Nickie L.; Rothfuss, Heather M.; Weisman, Michael H.; Solomon, Joshua J.; Fischer, Aryeh; Okamoto, Yuko; Kelmenson, Lindsay B.; Parish, Mark C.; Feser, Marie; Fleischer, Chelsie; Anderson, Courtney; Mahler, Michael; Norris, Jill M.; Kaplan, Mariana J.; Cherrington, Brian D.; Holers, V. Michael; Deane, Kevin D.

2017-01-01

Objectives Studies suggest that rheumatoid arthritis (RA)-related autoimmunity is initiated at a mucosal site. However, the factors associated with the mucosal generation of this autoimmunity are unknown, especially in individuals who are at-risk for future RA. Therefore, we tested anti-cyclic citrullinated peptide (anti-CCP) antibodies in the sputum of RA-free first-degree relatives (FDRs) of RA patients and patients with classifiable RA. Methods We evaluated induced sputum and serum from 67 FDRs and 20 RA subjects for anti-CCP-IgA and anti-CCP-IgG, with cut-off levels for positivity determined in a control population. Sputum was also evaluated for cell counts, neutrophil extracellular traps (NETs) using sandwich ELISAs for protein/nucleic acid complexes, and total citrulline. Results Sputum anti-CCP-IgA and/or anti-CCP-IgG was positive in 17/67 (25%) FDRs and 14/20 (70%) RA subjects, including a portion of FDRs who were serum anti-CCP negative. In FDRs, elevations of sputum anti-CCP-IgA and anti-CCP-IgG were associated with elevated sputum cell counts and levels of NET complexes. Anti-CCP-IgA was associated with ever-smoking and elevated sputum citrulline levels. Conclusions Anti-CCP is elevated in the sputum of FDRs, including seronegative FDRs, suggesting the lung may be one site of anti-CCP generation in this population. The association of anti-CCP with elevated cell counts and NET levels in FDRs supports a hypothesis that local airway inflammation and NET formation may drive anti-CCP production in the lung and may promote the early stages of RA development. Longitudinal studies are needed to follow the evolution of these processes relative to the development of systemic autoimmunity and articular RA. PMID:28182854

Effects of prednisone on eosinophilic bronchitis in asthma: a systematic review and meta-analysis*,**

PubMed Central

Sakae, Thiago Mamôru; Maurici, Rosemeri; Trevisol, Daisson José; Pizzichini, Marcia Margaret Menezes; Pizzichini, Emílio

2014-01-01

OBJECTIVE: To evaluate the effect size of oral corticosteroid treatment on eosinophilic bronchitis in asthma, through systematic review and meta-analysis. METHODS: We systematically reviewed articles in the Medline, Cochrane Controlled Trials Register, EMBASE, and LILACS databases. We selected studies meeting the following criteria: comparing at least two groups or time points (prednisone vs. control, prednisone vs. another drug, or pre- vs. post-treatment with prednisone); and evaluating parameters before and after prednisone use, including values for sputum eosinophils, sputum eosinophil cationic protein (ECP), and sputum IL-5-with or without values for post-bronchodilator FEV1-with corresponding 95% CIs or with sufficient data for calculation. The independent variables were the use, dose, and duration of prednisone treatment. The outcomes evaluated were sputum eosinophils, IL-5, and ECP, as well as post-bronchodilator FEV1. RESULTS: The pooled analysis of the pre- vs. post-treatment data revealed a significant mean reduction in sputum eosinophils (↓8.18%; 95% CI: 7.69-8.67; p < 0.001), sputum IL-5 (↓83.64 pg/mL; 95% CI: 52.45-114.83; p < 0.001), and sputum ECP (↓267.60 µg/L; 95% CI: 244.57-290.63; p < 0.0001), as well as a significant mean increase in post-bronchodilator FEV1 (↑8.09%; 95% CI: 5.35-10.83; p < 0.001). CONCLUSIONS: In patients with moderate-to-severe eosinophilic bronchitis, treatment with prednisone caused a significant reduction in sputum eosinophil counts, as well as in the sputum levels of IL-5 and ECP. This reduction in the inflammatory response was accompanied by a significant increase in post-bronchodilator FEV1. PMID:25410844

Factors Affecting Time to Sputum Culture Conversion in Adults with Pulmonary Tuberculosis: A Historical Cohort Study without Censored Cases.

PubMed

Kanda, Rie; Nagao, Taishi; Tho, Nguyen Van; Ogawa, Emiko; Murakami, Yoshitaka; Osawa, Makoto; Saika, Yoshinori; Doi, Kenji; Nakano, Yasutaka

2015-01-01

In patients with pulmonary tuberculosis (TB), shortening the time to sputum culture conversion is desirable to reduce the likelihood of mycobacterial transmission. A persistent positive sputum culture after 2 months of treatment is reported to be associated with the presence of cavitation and the extent of disease on chest X-ray, high colony count, diabetes mellitus, and smoking. However, little is known about factors affecting the time to sputum culture conversion. This study was conducted to evaluate factors affecting the time to sputum culture conversion throughout the course of treatment in adults with pulmonary TB. This study was performed using a database of the medical records of patients with active pulmonary TB who were treated at Hirakata Kohsai Hospital in Hirakata City, Osaka, Japan, from October 2000 to October 2002. Cox proportional-hazards analysis was used to evaluate factors affecting the time to sputum culture conversion after adjusting for potential confounders. The data of 86 patients with pulmonary TB were analyzed. The median time to sputum culture conversion was 39 days, and the maximum time was 116 days. The Cox proportional-hazards analysis showed that a higher smear grading (HR, 0.40; 95%CI, 0.23-0.71) and a history of ever smoking (HR, 0.48; 95%CI, 0.25-0.94) were associated with delayed sputum culture conversion. High smear grading and smoking prolonged the time to sputum culture conversion in adults with pulmonary TB. To effectively control TB, measures to decrease the cigarette smoking rate should be implemented, in addition to early detection and timely anti-TB treatment.

Comparison of biomarkers in serum and induced sputum of patients with occupational asthma and chronic obstructive pulmonary disease.

PubMed

Kleniewska, Aneta; Walusiak-Skorupa, Jolanta; Piotrowski, Wojciech; Nowakowska-Świrta, Ewa; Wiszniewska, Marta

2016-07-22

Occupational asthma and chronic obstructive pulmonary disease (COPD) are associated with the airway inflammatory process. The aim of this study was to compare the sputum and serum markers of inflammation in patients with occupational asthma and COPD. The study group included 20 patients with stable COPD, 24 patients with asthma, and 22 healthy subjects. Interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-9 levels in serum and induced sputum as well as fibrinogen and CRP in serum were determined in all the subjects. Higher concentrations of IL-1β, IL-6, TNF-α, and MMP-9 in induced sputum and an increased concentration of acute-phase proteins in serum were observed in COPD patients compared with healthy subjects. Higher concentrations of IL-1β and MMP-9 in induced sputum and a higher concentration of C-reactive protein (CRP) were detected in COPD patients than in asthmatic subjects. Never smokers with COPD had significantly higher levels of IL-1β and MMP-9 in induced sputum than never smoker controls. There was no significant difference between the serum and sputum levels of cytokines and MMP-9 of never smokers and smokers with COPD. Higher concentrations of IL-1β and MMP-9 in induced sputum and a higher concentration of CRP in serum allow distinguishing between biomarker profiles of COPD patients and asthmatic patients. Occupational exposure induces a systemic proinflammatory state with increased levels of acute-phase proteins in stable COPD patients. MMP-9 and IL-1β concentrations are increased in induced sputum of never smokers with COPD, which is associated with occupational exposure.

THE VIABILITY OF THE PNEUMOCOCCUS AFTER DRYING: A STUDY OF ONE OF THE FACTORS IN PNEUMONIC INFECTION

PubMed Central

Wood, Francis Carter

1905-01-01

I. In moist sputum kept in the dark at room temperatures the average life of the pneumococcus is eleven days, though considerable variations may be noted in different specimens of sputum. In the same sputum kept at o° C. the average life of the organism is thirty-five days. In sputum kept at room temperature and in a strong light the pneumococcus lives less than five days. II. In dried sputum (a) in the dark the pneumococcus lives on an average thirty-five days; (b) in diffuse light, thirty days; (c) in sunlight, less than four hours. III. In powdered sputum even when kept in the dark the death of the pneumococcus takes place in from one to four hours. When exposed to sunlight death occurs within an hour. IV. No important differences were noted in the life of the pneumococcus when dried on glass, tin, or wood. On cloth the life was usually slightly longer than on non-absorbing surfaces. V. Sprayed sputum particles remain in suspension for twenty-four hours, but all masses of a size sufficient to contain bacteria settle at a rate of about 40 cm. per hour. VI. When sputum containing pneumococci is sprayed the organisms rarely survive for more than an hour, and often die in less time. The substance upon which the particles fall makes but little difference in the life of the organism. On cloth a slight prolongation is occasionally noted, due perhaps to the slow drying. VII. The mucus of the sputum exerts a destructive action on the pneumococcus. VIII. Exposure of bacterial spray to sunlight while in suspension results in the destruction of the pneumococcus within half an hour. PMID:19867014

Effects of TiO{sub 2} addition on ionic conductivity of PVC/PEMA blend based composite polymer electrolyte

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subban, R. H. Y.; Facultyof Applied Sciences Universiti Teknologi MARA40450 Shah Alam Selangor Malaysia; Sukri, Nursyazwani

2015-08-28

PVC/PEMA blend based polymer electrolytes with lithium bistrifluoromethane sulfonimide (LiN(CF{sub 3}SO{sub 2}){sub 2}) and PVC/PEMA/(LiN(CF{sub 3}SO{sub 2}){sub 2}-TiO{sub 2} films were prepared by solution cast technique. The sample containing 35 wt. % LiN(CF{sub 3}SO{sub 2}){sub 2} exhibited the highest conductivity of 1.75 × 10{sup −5} Scm{sup −1}. The conductivity of the sample increased to 2.12 × 10{sup −5} Scm{sup −1} and 4.61 × 10{sup −5} Scm{sup −1} when 4 wt. % and 10 wt. % of titanium dioxide (TiO{sub 2}) was added to the sample at 65 wt. % PVC/PEMA-35 wt. % LiN(CF{sub 3}SO{sub 2}){sub 2} composition respectively. The lowmore » increase in conductivity is attributed to two competing factors: increase in crystallinity as accounted by X-Ray diffraction (XRD) and decrease in glass transition temperature as accounted by differential scanning calorimetry (DSC)« less

Tectonic activity as a significant source of crustal tetrafluoromethane emissions to the atmosphere: observations in groundwaters along the San Andreas Fault

USGS Publications Warehouse

Deeds, Daniel A.; Kulongoski, Justin T.; Muhle, Jens; Weiss, Ray F.

2015-01-01

Tetrafluoromethane (CF4) concentrations were measured in 14 groundwater samples from the Cuyama Valley, Mil Potrero and Cuddy Valley aquifers along the Big Bend section of the San Andreas Fault System (SAFS) in California to assess whether tectonic activity in this region is a significant source of crustal CF4 to the atmosphere. Dissolved CF4 concentrations in all groundwater samples but one were elevated with respect to estimated recharge concentrations including entrainment of excess air during recharge (CreCre; ∼30 fmol kg−1 H2O), indicating subsurface addition of CF4 to these groundwaters. Groundwaters in the Cuyama Valley contain small CF4 excesses (0.1–9 times CreCre), which may be attributed to an in situ release from weathering and a minor addition of deep crustal CF4 introduced to the shallow groundwater through nearby faults. CF4 excesses in groundwaters within 200 m of the SAFS are larger (10–980 times CreCre) and indicate the presence of a deep crustal flux of CF4 that is likely associated with the physical alteration of silicate minerals in the shear zone of the SAFS. Extrapolating CF4 flux rates observed in this study to the full extent of the SAFS (1300 km × 20–100 km) suggests that the SAFS potentially emits (0.3–1)×10−1 kg(0.3–1)×10−1 kg CF4 yr−1 to the Earth's surface. For comparison, the chemical weathering of ∼7.5×104 km2∼7.5×104 km2 of granitic rock in California is estimated to release (0.019–3.2)×10−1 kg(0.019–3.2)×10−1 kg CF4 yr−1. Tectonic activity is likely an important, and potentially the dominant, driver of natural emissions of CF4 to the atmosphere. Variations in preindustrial atmospheric CF4 as observed in paleo-archives such as ice cores may therefore represent changes in both continental weathering and tectonic activity, including changes driven by variations in continental ice cover during glacial–interglacial transitions.

Lung cancer, brucellosis and tuberculosis: remarkable togetherness

PubMed Central

Akkoyunlu, Muhammed Emin; Akkoyunlu, Yasemin; Hakyemez, Ismail Necati; Erboy, Fatma; Arvas, Gulhan; Aslan, Turan

2013-01-01

A 68 years old male farmer referred with cough, expectorating sputum, intermittant fever, night sweats, fatigue and anorexia persisting for two weeks. There was a history of 80 packs each year of smoking and he was still an active smoker. Pneumonectomy was performed because of pulmonary epidermoid cancer and he received chemotherapy. He was diagnosed lung tuberculosis and using anti-tuberculous treatment for 4 months. He had a weight loss of 8 kg in last month. His body tempereature was 38.5 °C. Heart rate was 100/min. ESR was 51mm/h and CRP was 5.6 mg/dL. There was no proliferation in blood and sputum cultures. Three sputum specimens were examined and AFB wasn't detected. Fibronodular infiltration was seen in right lower zone of chest X-ray. In thorax CT, fibronodular densities were seen in lower lobe anterior and posterior segments. Brucella melitensis was isolated in blood culture. Second bronchoscopy was performed with suspect of brucellosis pneumonia. Brucella tube agglutination test was positive at titer 1/320 in the bronchial lavage fluid and 1/640 in concurrent serum sample. In cases with chronic cough or pneumonia which is irresponsive to nonspecific antibiotherapy, respiratory brucellosis must be rememberred in endemic areas.

Preliminary investigation of human exhaled breath for tuberculosis diagnosis by multidimensional gas chromatography - Time of flight mass spectrometry and machine learning.

PubMed

Beccaria, Marco; Mellors, Theodore R; Petion, Jacky S; Rees, Christiaan A; Nasir, Mavra; Systrom, Hannah K; Sairistil, Jean W; Jean-Juste, Marc-Antoine; Rivera, Vanessa; Lavoile, Kerline; Severe, Patrice; Pape, Jean W; Wright, Peter F; Hill, Jane E

2018-02-01

Tuberculosis (TB) remains a global public health malady that claims almost 1.8 million lives annually. Diagnosis of TB represents perhaps one of the most challenging aspects of tuberculosis control. Gold standards for diagnosis of active TB (culture and nucleic acid amplification) are sputum-dependent, however, in up to a third of TB cases, an adequate biological sputum sample is not readily available. The analysis of exhaled breath, as an alternative to sputum-dependent tests, has the potential to provide a simple, fast, and non-invasive, and ready-available diagnostic service that could positively change TB detection. Human breath has been evaluated in the setting of active tuberculosis using thermal desorption-comprehensive two-dimensional gas chromatography-time of flight mass spectrometry methodology. From the entire spectrum of volatile metabolites in breath, three random forest machine learning models were applied leading to the generation of a panel of 46 breath features. The twenty-two common features within each random forest model used were selected as a set that could distinguish subjects with confirmed pulmonary M. tuberculosis infection and people with other pathologies than TB. Copyright © 2018 Elsevier B.V. All rights reserved.

A sensitive method to quantify human cell-free circulating DNA in blood: relevance to myocardial infarction screening.

PubMed

Jing, Rong-Rong; Wang, Hui-Min; Cui, Ming; Fang, Meng-Kang; Qiu, Xiao-Jun; Wu, Xin-Hua; Qi, Jin; Wang, Yue-Guo; Zhang, Lu-Rong; Zhu, Jian-Hua; Ju, Shao-Qing

2011-09-01

Human cell-free circulating DNA (cf-DNA) derived mainly from cell apoptosis and necrosis can be measured by a variety of laboratory techniques, but almost all of these methods require sample preparation. We have developed a branched DNA (bDNA)-based Alu assay for quantifying cf-DNA in myocardial infarction (MI) patients. A total of 82 individuals were included in the study; 22 MI and 60 normal controls. cf-DNA was quantified using a bDNA-based Alu assay. cf-DNA was higher in serum compared to plasma and there was a difference between genders. cf-DNA was significantly higher in MI patients compared to the controls. There was no correlation between cf-DNA and creatine kinase-MB (CK-MB), troponin I (cTnI) or myoglobin (MYO). In serial specimens, cf-DNA was sensitive and peaked earlier than cTnI. The bDNA-based Alu assay is a novel method for quantifying human cf-DNA. Increased cf-DNA in MI patients might complement cTnI, CK-MB and MYO in a multiple marker format. Copyright © 2011 The Canadian Society of Clinical Chemists. All rights reserved.

KRAS mutations in blood circulating cell-free DNA: a pancreatic cancer case-control

PubMed Central

Le Calvez-Kelm, Florence; Foll, Matthieu; Wozniak, Magdalena B.; Delhomme, Tiffany M.; Durand, Geoffroy; Chopard, Priscilia; Pertesi, Maroulio; Fabianova, Eleonora; Adamcakova, Zora; Holcatova, Ivana; Foretova, Lenka; Janout, Vladimir; Vallee, Maxime P.; Rinaldi, Sabina; Brennan, Paul; McKay, James D.; Byrnes, Graham B.; Scelo, Ghislaine

2016-01-01

The utility of KRAS mutations in plasma circulating cell-free DNA (cfDNA) samples as non-invasive biomarkers for the detection of pancreatic cancer has never been evaluated in a large case-control series. We applied a KRAS amplicon-based deep sequencing strategy combined with analytical pipeline specifically designed for the detection of low-abundance mutations to screen plasma samples of 437 pancreatic cancer cases, 141 chronic pancreatitis subjects, and 394 healthy controls. We detected mutations in 21.1% (N=92) of cases, of whom 82 (89.1%) carried at least one mutation at hotspot codons 12, 13 or 61, with mutant allelic fractions from 0.08% to 79%. Advanced stages were associated with an increased proportion of detection, with KRAS cfDNA mutations detected in 10.3%, 17,5% and 33.3% of cases with local, regional and systemic stages, respectively. We also detected KRAS cfDNA mutations in 3.7% (N=14) of healthy controls and in 4.3% (N=6) of subjects with chronic pancreatitis, but at significantly lower allelic fractions than in cases. Combining cfDNA KRAS mutations and CA19-9 plasma levels on a limited set of case-control samples did not improve the overall performance of the biomarkers as compared to CA19-9 alone. Whether the limited sensitivity and specificity observed in our series of KRAS mutations in plasma cfDNA as biomarkers for pancreatic cancer detection are attributable to methodological limitations or to the biology of cfDNA should be further assessed in large case-control series. PMID:27705932

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy).

PubMed

Garcia, Jessica; Forestier, Julien; Dusserre, Eric; Wozny, Anne-Sophie; Geiguer, Florence; Merle, Patrick; Tissot, Claire; Ferraro-Peyret, Carole; Jones, Frederick S; Edelstein, Daniel L; Cheynet, Valérie; Bardel, Claire; Vilchez, Gaelle; Xu, Zhenyu; Bringuier, Pierre Paul; Barritault, Marc; Brengle-Pesce, Karen; Guillet, Marielle; Chauvenet, Marion; Manship, Brigitte; Brevet, Marie; Rodriguez-Lafrasse, Claire; Hervieu, Valérie; Couraud, Sébastien; Walter, Thomas; Payen, Léa

2018-04-20

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5-1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results.

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy)

PubMed Central

Garcia, Jessica; Forestier, Julien; Dusserre, Eric; Wozny, Anne-Sophie; Geiguer, Florence; Merle, Patrick; Tissot, Claire; Ferraro-Peyret, Carole; Jones, Frederick S.; Edelstein, Daniel L.; Cheynet, Valérie; Bardel, Claire; Vilchez, Gaelle; Xu, Zhenyu; Bringuier, Pierre Paul; Barritault, Marc; Brengle-Pesce, Karen; Guillet, Marielle; Chauvenet, Marion; Manship, Brigitte; Brevet, Marie; Rodriguez-Lafrasse, Claire; Hervieu, Valérie; Couraud, Sébastien; Walter, Thomas; Payen, Léa

2018-01-01

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5–1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results. PMID:29765524

Discovery and Validation of a Six-Marker Serum Protein Signature for the Diagnosis of Active Pulmonary Tuberculosis.

PubMed

De Groote, Mary A; Sterling, David G; Hraha, Thomas; Russell, Theresa M; Green, Louis S; Wall, Kirsten; Kraemer, Stephan; Ostroff, Rachel; Janjic, Nebojsa; Ochsner, Urs A

2017-10-01

New non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant ( P < 10 -20 ) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform. Copyright © 2017 De Groote et al.

Discovery and Validation of a Six-Marker Serum Protein Signature for the Diagnosis of Active Pulmonary Tuberculosis

PubMed Central

De Groote, Mary A.; Sterling, David G.; Hraha, Thomas; Russell, Theresa M.; Green, Louis S.; Wall, Kirsten; Kraemer, Stephan; Ostroff, Rachel; Janjic, Nebojsa

2017-01-01

ABSTRACT New non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant (P < 10−20) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform. PMID:28794177

Analysis of contributing factors associated to related patients safety incidents in Intensive Care Medicine.

PubMed

Martín Delgado, M C; Merino de Cos, P; Sirgo Rodríguez, G; Álvarez Rodríguez, J; Gutiérrez Cía, I; Obón Azuara, B; Alonso Ovies, Á

2015-01-01

To explore contributing factors (CF) associated to related critical patients safety incidents. SYREC study pos hoc analysis. A total of 79 Intensive Care Departments were involved. The study sample consisted of 1.017 patients; 591 were affected by one or more incidents. The CF were categorized according to a proposed model by the National Patient Safety Agency from United Kingdom that was modified. Type, class and severity of the incidents was analyzed. A total 2,965 CF were reported (1,729 were associated to near miss and 1,236 to adverse events). The CF group more frequently reported were related patients factors. Individual factors were reported more frequently in near miss and task related CF in adverse events. CF were reported in all classes of incidents. The majority of CF were reported in the incidents classified such as less serious, even thought CF patients factors were associated to serious incidents. Individual factors were considered like avoidable and patients factors as unavoidable. The CF group more frequently reported were patient factors and was associated to more severe and unavoidable incidents. By contrast, individual factors were associated to less severe and avoidable incidents. In general, CF most frequently reported were associated to near miss. Copyright © 2014 Elsevier España, S.L.U. and SEMICYUC. All rights reserved.

A comparison of sputum induction methods: ultrasonic vs compressed-air nebulizer and hypertonic vs isotonic saline inhalation.

PubMed

Loh, L C; Eg, K P; Puspanathan, P; Tang, S P; Yip, K S; Vijayasingham, P; Thayaparan, T; Kumar, S

2004-03-01

Airway inflammation can be demonstrated by the modem method of sputum induction using ultrasonic nebulizer and hypertonic saline. We studied whether compressed-air nebulizer and isotonic saline which are commonly available and cost less, are as effective in inducing sputum in normal adult subjects as the above mentioned tools. Sixteen subjects underwent weekly sputum induction in the following manner: ultrasonic nebulizer (Medix Sonix 2000, Clement Clarke, UK) using hypertonic saline, ultrasonic nebulizer using isotonic saline, compressed-air nebulizer (BestNeb, Taiwan) using hypertonic saline, and compressed-air nebulizer using isotonic saline. Overall, the use of an ultrasonic nebulizer and hypertonic saline yielded significantly higher total sputum cell counts and a higher percentage of cell viability than compressed-air nebulizers and isotonic saline. With the latter, there was a trend towards squamous cell contaminations. The proportion of various sputum cell types was not significantly different between the groups, and the reproducibility in sputum macrophages and neutrophils was high (Intraclass correlation coefficient, r [95%CI]: 0.65 [0.30-0.91] and 0.58 [0.22-0.89], p < 0.001). Overall changes in median FEV, were small and comparable between all groups. Induction using ultrasonic nebulizers together with hypertonic saline was generally less well tolerated than compressed-air nebulizers and isotonic saline. We conclude that in normal subjects, although both nebulizers and saline types can induce sputum with reproducible cellular profile, ultrasonic nebulizers and hypertonic saline are more effective but less well tolerated.

Identification of mycobacterium tuberculosis in sputum smear slide using automatic scanning microscope

NASA Astrophysics Data System (ADS)

Rulaningtyas, Riries; Suksmono, Andriyan B.; Mengko, Tati L. R.; Saptawati, Putri

2015-04-01

Sputum smear observation has an important role in tuberculosis (TB) disease diagnosis, because it needs accurate identification to avoid high errors diagnosis. In development countries, sputum smear slide observation is commonly done with conventional light microscope from Ziehl-Neelsen stained tissue and it doesn't need high cost to maintain the microscope. The clinicians do manual screening process for sputum smear slide which is time consuming and needs highly training to detect the presence of TB bacilli (mycobacterium tuberculosis) accurately, especially for negative slide and slide with less number of TB bacilli. For helping the clinicians, we propose automatic scanning microscope with automatic identification of TB bacilli. The designed system modified the field movement of light microscope with stepper motor which was controlled by microcontroller. Every sputum smear field was captured by camera. After that some image processing techniques were done for the sputum smear images. The color threshold was used for background subtraction with hue canal in HSV color space. Sobel edge detection algorithm was used for TB bacilli image segmentation. We used feature extraction based on shape for bacilli analyzing and then neural network classified TB bacilli or not. The results indicated identification of TB bacilli that we have done worked well and detected TB bacilli accurately in sputum smear slide with normal staining, but not worked well in over staining and less staining tissue slide. However, overall the designed system can help the clinicians in sputum smear observation becomes more easily.

Noninvasive analysis of the sputum transcriptome discriminates clinical phenotypes of asthma.

PubMed

Yan, Xiting; Chu, Jen-Hwa; Gomez, Jose; Koenigs, Maria; Holm, Carole; He, Xiaoxuan; Perez, Mario F; Zhao, Hongyu; Mane, Shrikant; Martinez, Fernando D; Ober, Carole; Nicolae, Dan L; Barnes, Kathleen C; London, Stephanie J; Gilliland, Frank; Weiss, Scott T; Raby, Benjamin A; Cohn, Lauren; Chupp, Geoffrey L

2015-05-15

The airway transcriptome includes genes that contribute to the pathophysiologic heterogeneity seen in individuals with asthma. We analyzed sputum gene expression for transcriptomic endotypes of asthma (TEA), gene signatures that discriminate phenotypes of disease. Gene expression in the sputum and blood of patients with asthma was measured using Affymetrix microarrays. Unsupervised clustering analysis based on pathways from the Kyoto Encyclopedia of Genes and Genomes was used to identify TEA clusters. Logistic regression analysis of matched blood samples defined an expression profile in the circulation to determine the TEA cluster assignment in a cohort of children with asthma to replicate clinical phenotypes. Three TEA clusters were identified. TEA cluster 1 had the most subjects with a history of intubation (P = 0.05), a lower prebronchodilator FEV1 (P = 0.006), a higher bronchodilator response (P = 0.03), and higher exhaled nitric oxide levels (P = 0.04) compared with the other TEA clusters. TEA cluster 2, the smallest cluster, had the most subjects that were hospitalized for asthma (P = 0.04). TEA cluster 3, the largest cluster, had normal lung function, low exhaled nitric oxide levels, and lower inhaled steroid requirements. Evaluation of TEA clusters in children confirmed that TEA clusters 1 and 2 are associated with a history of intubation (P = 5.58 × 10(-6)) and hospitalization (P = 0.01), respectively. There are common patterns of gene expression in the sputum and blood of children and adults that are associated with near-fatal, severe, and milder asthma.

15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis.

PubMed

Profita, M; Sala, A; Riccobono, L; Pace, E; Paternò, A; Zarini, S; Siena, L; Mirabella, A; Bonsignore, G; Vignola, A M

2000-10-01

We evaluated the levels of 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of 15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 control and 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reverse phase high-performance liquid chromatography separation followed by specific RIA. 15-LO mRNA expression was determined by primed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than in control subjects. The percentage of cells expressing 15-LO mRNA was significantly higher in chronic bronchitis than in control subjects (P < 0.01). Double staining for specific cell type markers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did not show evidence for 15-LO expression, suggesting that expression of 15-LO in neutrophils takes place on migration into the airways. Because 15(S)-HETE inversely correlated with the percentage of neutrophils in sputum of chronic bronchitis subjects, we studied the effect of 15(S)-HETE on leukotriene B(4) (LTB(4)) production in vitro and evaluated the concentration of LTB(4) in induced sputum and the contribution of LTB(4) to the chemotactic activity of induced sputum samples ex vivo. The results obtained indicate that macrophages and neutrophils present within the airways of chronic bronchitis subjects express 15-LO mRNA; increased basal levels of 15(S)-HETE may contribute to modulate, through the inhibition of 5-lipoxygenase metabolites production, neutrophil infiltration and airway inflammation associated with chronic bronchitis.

Noninvasive Analysis of the Sputum Transcriptome Discriminates Clinical Phenotypes of Asthma

PubMed Central

Yan, Xiting; Chu, Jen-Hwa; Gomez, Jose; Koenigs, Maria; Holm, Carole; He, Xiaoxuan; Perez, Mario F.; Zhao, Hongyu; Mane, Shrikant; Martinez, Fernando D.; Ober, Carole; Nicolae, Dan L.; Barnes, Kathleen C.; London, Stephanie J.; Gilliland, Frank; Weiss, Scott T.; Raby, Benjamin A.; Cohn, Lauren

2015-01-01

Rationale: The airway transcriptome includes genes that contribute to the pathophysiologic heterogeneity seen in individuals with asthma. Objectives: We analyzed sputum gene expression for transcriptomic endotypes of asthma (TEA), gene signatures that discriminate phenotypes of disease. Methods: Gene expression in the sputum and blood of patients with asthma was measured using Affymetrix microarrays. Unsupervised clustering analysis based on pathways from the Kyoto Encyclopedia of Genes and Genomes was used to identify TEA clusters. Logistic regression analysis of matched blood samples defined an expression profile in the circulation to determine the TEA cluster assignment in a cohort of children with asthma to replicate clinical phenotypes. Measurements and Main Results: Three TEA clusters were identified. TEA cluster 1 had the most subjects with a history of intubation (P = 0.05), a lower prebronchodilator FEV1 (P = 0.006), a higher bronchodilator response (P = 0.03), and higher exhaled nitric oxide levels (P = 0.04) compared with the other TEA clusters. TEA cluster 2, the smallest cluster, had the most subjects that were hospitalized for asthma (P = 0.04). TEA cluster 3, the largest cluster, had normal lung function, low exhaled nitric oxide levels, and lower inhaled steroid requirements. Evaluation of TEA clusters in children confirmed that TEA clusters 1 and 2 are associated with a history of intubation (P = 5.58 × 10−6) and hospitalization (P = 0.01), respectively. Conclusions: There are common patterns of gene expression in the sputum and blood of children and adults that are associated with near-fatal, severe, and milder asthma. PMID:25763605

VizieR Online Data Catalog: Radial velocities in M67. I. 1278 candidate members (Geller+, 2015)

NASA Astrophysics Data System (ADS)

Geller, A. M.; Latham, D. W.; Mathieu, R. D.

2015-10-01

This is the first in a series of papers studying the dynamical state of the old open cluster M67 through precise radial velocities. This is also the paper LXVII of the WIYN Open Cluster Study. Our radial velocity survey of M67 began as part of the dissertation work of Mathieu (1983PhDT.........8M), taking advantage of the CfA Digital Speedometers (DS). Three nearly identical instruments were used, initially on the MMT (from HJD2445337 to HJD2450830) and 1.5m Tillinghast Reflector (from HJD2444184 to HJD2454958) at the Fred Lawrence Whipple Observatory on Mount Hopins, Arizona, and then later on the 1.5m Wyeth Reflector (from HJD2445722 to HJD2453433) at the Oak Ridge Observatory in the Town of Harvard, Massachusetts. Subsequently the M67 target samples were expanded several times. Radial velocities measurements from other programs were integrated into the database, and our observational facilities were extended to include Hydra Multi-Object Spectrograph (MOS) at the WIYN Observatory (from HJD2453386 to HJD2456709) and the new Tillinghast Reflector Echelle Spectrograph (TRES) on the Tillinghast Reflector (from HJD2455143 to HJD2456801). Details about the telescopes, observing procedures, and data reductions of spectra obtained with the CfA DS can be found in Latham (1985srv..conf...21L, 1992ASPC...32..110L). The corresponding information for spectra obtained with Hydra at the WIYN Observatory can be found in Geller et al. 2008 (cat. J/AJ/135/2264), Geller et al. 2010 (cat. J/AJ/139/1383) and Hole et al. (2009). TRES is a stabilized fiber-fed echelle spectrograph with a CCD detector and resolution of 44000. The initial CfA sample was defined in 1982. The last surviving CfA Digital Speedometer, on the 1.5m Tillinghast Reflector, was retired in the summer of 2009. Over the following five observing seasons, TRES was used to continue the radial velocity observations of targets (mostly binaries) from both the CfA and the WIYN samples. Importantly, Roger Griffin and James Gunn also had a radial velocity program for M67 from 1971 to 1982 at the Palomar Hale 5m telescope (from HJD2440952 to HJD2445297), which they supplemented with contemporaneous observations obtained with the CORAVEL instrument (from HJD2444340 to HJD2446413) at Haute Provence for five of the binaries. Their target sample was very similar to our initial sample. We refer to the target stars and the measurements taken at all telescope/instrument pairs except WIYN/Hydra as the CfA sample and data. As of 2015 February, there are 447 stars in the CfA sample. WIYN observations of M67 began on 2005 January 15 as part of the WIYN Open Cluster Study (WOCS; Mathieu, 2000ASPC..198..517M). In total there are 1278 stars within the WIYN stellar sample; 382 of these stars are also in the CfA stellar sample. (1 data file).

Volume dependency for culture of fungi from respiratory secretions and increased sensitivity of Aspergillus quantitative PCR.

PubMed

Fraczek, Marcin G; Kirwan, Marie B; Moore, Caroline B; Morris, Julie; Denning, David W; Richardson, Malcolm D

2014-02-01

Diagnosis of aspergillosis is often difficult. We compared fungal yields from respiratory specimens using the Health Protection Agency standard culture method (BSOP57), a higher volume undiluted culture method Mycology Reference Centre Manchester (MRCM) and Aspergillus quantitative real time polymerase chain reaction (qPCR). Sputum, bronchial aspirate and bronchoalveolar lavage (BAL) samples (total 23) were collected from aspergillosis patients. One fraction of all samples was cultured using the MRCM method, one BSOP57 and one was used for qPCR. The recovery rate for fungi was significantly higher by MRCM (87%) than by BSOP57 (8.7%) from all 23 specimens. Sputum samples were 44% positive by MRCM compared to no fungi isolated (0%) by BSOP57. Bronchial aspirates were 75% positive by MRCM and 0% by BSOP57. BAL samples were positive in 20% by MRCM and 10% by BSOP57. qPCR was always more sensitive than culture (95.6%) from all samples. In general, over 100 mould colonies (81 Aspergillus fumigatus) were grown using the MRCM method compared with only one colony from BSOP57. This study provides a reference point for standardisation of respiratory sample processing in diagnostic laboratories. Culture from higher volume undiluted respiratory specimens has a much higher yield for Aspergillus than BSOP57. qPCR is much more sensitive than culture and the current UK method requires revision. © 2013 Blackwell Verlag GmbH.

Cold flow of estradiol transdermal systems: influence of drug loss on the in vitro flux and drug transfer across human epidermis.

PubMed

Krishnaiah, Yellela S R; Yang, Yang; Hunt, Robert L; Khan, Mansoor A

2014-12-30

The objective was to quantify drug loss due to cold flow (CF) in marketed estradiol transdermal drug delivery systems (TDDS), and study its influence on the in vitro flux and drug transfer across contacting skin. TDDS samples (products-A and B) were induced with CF at 25 and 32°C/60% RH by applying 1-kg force for 72h. CF was measured as percent dimensional change and amount of drug loss/migration in CF region. In vitro drug permeation studies were conducted across human epidermis from TDDS excluding CF region, and CF region alone against control (without CF). In both products, significantly higher percentage of CF (dimensional change and drug migration) was observed at 32°C compared to 25°C. In vitro flux from both products excluding CF region either at 25 or 32°C was the same, but significantly lower compared to control. Drug transferred from CF region of product-A after 8h was the same at 25 and 32°C, but significantly higher in product-B. Flux from both products together with CF region at 32°C was significantly lower than that observed at 25°C. Results showed that excessive CF at storage (25°C) and clinical usage (32°C) conditions may have implications on product performance and safety of estradiol TDDS. Published by Elsevier B.V.

Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**

EPA Science Inventory

Rationale: The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. ...

Pepsin and bile acid concentrations in sputum of mustard gas exposed patients.

PubMed

Karbasi, Ashraf; Goosheh, Hassan; Aliannejad, Rasoul; Saber, Hamid; Salehi, Maryam; Jafari, Mahvash; Imani, Saber; Saburi, Amin; Ghanei, Mostafa

2013-01-01

Gastro-esophageal reflux has been suggested to be associated with several pulmonary complications such as asthma, and post-transplant bronchiolitis obliterans (BO). Pepsin or bile salts in the sputum is shown to be an optimal molecular marker of gastric contents macro/micro aspiration. In this study, we investigated sputum pepsin as a marker of micro-aspiration in sulfur mustard (SM) exposed cases compared to healthy controls. In a case controlled study, 26 cases with BO and 12 matched healthy controls were recruited and all cases were symptomatic and their exposure to SM was previously documented during Iran-Iraq conflict. Pepsin levels in sputum and total bile acids were measured using enzymatic assay. The severity of respiratory disorder was categorized based upon the spirometric values. The average concentration of pepsin in sputum was higher in the case group (0.29 ± 0.23) compared with healthy subjects (0.13 ± 0.07; P ± 0.003). Moreover, the average concentration of bile acids in the sputum cases was not significantly different in comparison to the controls ( P = 0.5). Higher pepsin concentrations in sputum of SM exposed patients compared with healthy control subjects indicate the occurrence of significantly more gastric micro-aspiration in SM exposed patients.

Pepsin and Bile Acid Concentrations in Sputum of Mustard Gas Exposed Patients

PubMed Central

Karbasi, Ashraf; Goosheh, Hassan; Aliannejad, Rasoul; Saber, Hamid; Salehi, Maryam; Jafari, Mahvash; Imani, Saber; Saburi, Amin; Ghanei, Mostafa

2013-01-01

Background/Aim: Gastro-esophageal reflux has been suggested to be associated with several pulmonary complications such as asthma, and post-transplant bronchiolitis obliterans (BO). Pepsin or bile salts in the sputum is shown to be an optimal molecular marker of gastric contents macro/micro aspiration. In this study, we investigated sputum pepsin as a marker of micro-aspiration in sulfur mustard (SM) exposed cases compared to healthy controls. Materials and Methods: In a case controlled study, 26 cases with BO and 12 matched healthy controls were recruited and all cases were symptomatic and their exposure to SM was previously documented during Iran-Iraq conflict. Pepsin levels in sputum and total bile acids were measured using enzymatic assay. The severity of respiratory disorder was categorized based upon the spirometric values. Result: The average concentration of pepsin in sputum was higher in the case group (0.29 ± 0.23) compared with healthy subjects (0.13 ± 0.07; P ± 0.003). Moreover, the average concentration of bile acids in the sputum cases was not significantly different in comparison to the controls (P = 0.5). Conclusion: Higher pepsin concentrations in sputum of SM exposed patients compared with healthy control subjects indicate the occurrence of significantly more gastric micro-aspiration in SM exposed patients. PMID:23680709

Clostridium difficile carriage in adult cystic fibrosis (CF); implications for patients with CF and the potential for transmission of nosocomial infection.

PubMed

Burke, D G; Harrison, M J; Fleming, C; McCarthy, M; Shortt, C; Sulaiman, I; Murphy, D M; Eustace, J A; Shanahan, F; Hill, C; Stanton, C; Rea, M C; Ross, R P; Plant, B J

2017-03-01

Clostridium difficile is an anaerobic Gram-positive, spore-forming, toxin-producing bacillus transmitted among humans through the faecal-oral route. Despite increasing carriage rates and the presence of C. difficile toxin in stool, patients with CF rarely appear to develop typical manifestations of C. difficile infection (CDI). In this study, we examined the carriage, toxin production, ribotype distribution and antibiotic susceptibility of C. difficile in a cohort of 60 adult patients with CF who were pre-lung transplant. C. difficile was detected in 50% (30/60) of patients with CF by culturing for the bacteria. C. difficile toxin was detected in 63% (19/30) of C. difficile-positive stool samples. All toxin-positive stool samples contained toxigenic C. difficile strains harbouring toxin genes, tcdA and tcdB. Despite the presence of C. difficile and its toxin in patient stool, no acute gastrointestinal symptoms were reported. Ribotyping of C. difficile strains revealed 16 distinct ribotypes (RT), 11 of which are known to be disease-causing including the hyper-virulent RT078. Additionally, strains RT002, RT014, and RT015, which are common in non-CF nosocomial infection were described. All strains were susceptible to vancomycin, metronidazole, fusidic acid and rifampicin. No correlation was observed between carriage of C. difficile or any characteristics of isolated strains and any recorded clinical parameters or treatment received. We demonstrate a high prevalence of hypervirulent, toxigenic strains of C. difficile in asymptomatic patients with CF. This highlights the potential role of asymptomatic patients with CF in nosocomial transmission of C. difficile. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Environmental allergies and respiratory morbidities in cystic fibrosis.

PubMed

Collaco, Joseph M; Morrow, Christopher B; Green, Deanna M; Cutting, Garry R; Mogayzel, Peter J

2013-09-01

Cystic fibrosis (CF) is characterized by recurrent respiratory infections and progressive lung disease. Although environmental factors account for 50% of the variation in CF lung function, few specific exposures have been identified. Studies using small study samples focusing on environmental allergies in CF have had inconsistent results. Our objective was to examine the role of environmental allergies in upper and lower respiratory tract morbidities in CF. A total of 1,321 subjects with CF were recruited through the U.S. CF Twin-Sibling Study. Questionnaires were used to determine the presence/absence of environmental allergies. Questionnaires, chart review, and U.S. CF Foundation Patient Registry data were used to track outcomes. Within the study sample 14% reported environmental allergies. Environmental allergies were associated with a higher risk of sinus disease (adjusted OR: 2.68; P < 0.001) and nasal polyps (adjusted OR: 1.74; P = 0.003). Environmental allergies were also associated with a more rapid decline in lung function (additional -1.1%/year; P = 0.001). However, allergies were associated with a later median age of acquisition of Pseudomonas aeruginosa (6.6 years vs. 4.4 years; log rank P = 0.027). The reported use of common allergy medications, anti-histamines and leukotriene inhibitors, did not alter the frequency of respiratory morbidities. Environmental allergies are associated with an increased risk of sinus disease and nasal polyps and a more rapid decline in CF lung function, but may have a protective effect against the acquisition of P. aeruginosa. Prospective studies are needed to confirm these associations which have implications for more aggressive management of allergies. Copyright © 2012 Wiley Periodicals, Inc.

Environmental Allergies and Respiratory Morbidities in Cystic Fibrosis

PubMed Central

Collaco, Joseph M.; Morrow, Christopher B.; Green, Deanna M.; Cutting, Garry R.; Mogayzel, Peter J.

2012-01-01

SUMMARY Background Cystic fibrosis (CF) is characterized by recurrent respiratory infections and progressive lung disease. Although environmental factors account for 50% of the variation in CF lung function, few specific exposures have been identified. Studies using small study samples focusing on environmental allergies in CF have had inconsistent results. Our objective was to examine the role of environmental allergies in upper and lower respiratory tract morbidities in CF. Methods A total of 1321 subjects with CF were recruited through the U.S. CF Twin-Sibling Study. Questionnaires were used to determine the presence/absence of environmental allergies. Questionnaires, chart review, and U.S. CF Foundation Patient Registry data were used to track outcomes. Results Within the study sample 14% reported environmental allergies. Environmental allergies were associated with a higher risk of sinus disease (adjusted OR: 2.68; p<0.001) and nasal polyps (adjusted OR: 1.74; p=0.003). Environmental allergies were also associated with a more rapid decline in lung function (additional −1.1%/year; p=0.001). However, allergies were associated with a later median age of acquisition of Pseudomonas aeruginosa (6.6 yo vs. 4.4 yo; log rank p=0.027). The reported use of common allergy medications, anti-histamines and leukotriene inhibitors, did not alter the frequency of respiratory morbidities. Conclusions Environmental allergies are associated with an increased risk of sinus disease and nasal polyps and a more rapid decline in CF lung function, but may have a protective effect against the acquisition of P. aeruginosa. Prospective studies are needed to confirm these associations which have implications for more aggressive management of allergies. PMID:23143815

A new approach to epigenome-wide discovery of non-invasive methylation biomarkers for colorectal cancer screening in circulating cell-free DNA using pooled samples.

PubMed

Gallardo-Gómez, María; Moran, Sebastian; Páez de la Cadena, María; Martínez-Zorzano, Vicenta Soledad; Rodríguez-Berrocal, Francisco Javier; Rodríguez-Girondo, Mar; Esteller, Manel; Cubiella, Joaquín; Bujanda, Luis; Castells, Antoni; Balaguer, Francesc; Jover, Rodrigo; De Chiara, Loretta

2018-01-01

Colorectal cancer is the fourth cause of cancer-related deaths worldwide, though detection at early stages associates with good prognosis. Thus, there is a clear demand for novel non-invasive tests for the early detection of colorectal cancer and premalignant advanced adenomas, to be used in population-wide screening programs. Aberrant DNA methylation detected in liquid biopsies, such as serum circulating cell-free DNA (cfDNA), is a promising source of non-invasive biomarkers. This study aimed to assess the feasibility of using cfDNA pooled samples to identify potential serum methylation biomarkers for the detection of advanced colorectal neoplasia (colorectal cancer or advanced adenomas) using microarray-based technology. cfDNA was extracted from serum samples from 20 individuals with no colorectal findings, 20 patients with advanced adenomas, and 20 patients with colorectal cancer (stages I and II). Two pooled samples were prepared for each pathological group using equal amounts of cfDNA from 10 individuals, sex-, age-, and recruitment hospital-matched. We measured the methylation levels of 866,836 CpG positions across the genome using the MethylationEPIC array. Pooled serum cfDNA methylation data meets the quality requirements. The proportion of detected CpG in all pools (> 99% with detection p value < 0.01) exceeded Illumina Infinium methylation data quality metrics of the number of sites detected. The differential methylation analysis revealed 1384 CpG sites (5% false discovery rate) with at least 10% difference in the methylation level between no colorectal findings controls and advanced neoplasia, the majority of which were hypomethylated. Unsupervised clustering showed that cfDNA methylation patterns can distinguish advanced neoplasia from healthy controls, as well as separate tumor tissue from healthy mucosa in an independent dataset. We also observed that advanced adenomas and stage I/II colorectal cancer methylation profiles, grouped as advanced neoplasia, are largely homogenous and clustered close together. This preliminary study shows the viability of microarray-based methylation biomarker discovery using pooled serum cfDNA samples as an alternative approach to tissue specimens. Our strategy sets an open door for deciphering new non-invasive biomarkers not only for colorectal cancer detection, but also for other types of cancers.

The Italian National External quality assessment program in molecular genetic testing: results of the VII round (2010-2011).

PubMed

Censi, F; Tosto, F; Floridia, G; Marra, M; Salvatore, M; Baffico, A M; Grasso, M; Melis, M A; Pelo, E; Radice, P; Ravani, A; Rosatelli, C; Resta, N; Russo, S; Seia, M; Varesco, L; Falbo, V; Taruscio, D

2013-01-01

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

Production and investigation of thin films of metal actinides (Pu, Am, Cm, Bk, Cf)

NASA Astrophysics Data System (ADS)

Radchenko, V. M.; Ryabinin, M. A.; Stupin, V. A.

2010-03-01

Under limited availability of transplutonium metals some special techniques and methods of their production have been developed that combine the process of metal reduction from a chemical compound and preparation of a sample for examination. In this situation the evaporation and condensation of metal onto a substrate becomes the only possible technology. Thin film samples of metallic 244Cm, 248Cm and 249Bk were produced by thermal reduction of oxides with thorium followed by deposition of the metals in the form of thin layers on tantalum substrates. For the production of 249Cf metal in the form of a thin layer the method of thermal reduction of oxide with lanthanum was used. 238Pu and 239Pu samples in the form of films were prepared by direct high temperature evaporation and condensation of the metal onto a substrate. For the production of 241Am films a gram sample of plutonium-241 metal was used containing about 18 % of americium at the time of production. Thermal decomposition of Pt5Am intermetallics in vacuum was used to produce americium metal with about 80% yield. Resistivity of the metallic 249Cf film samples was found to decrease exponentially with increasing temperature. The 249Cf metal demonstrated a tendency to form preferably a DHCP structure with the sample mass increasing. An effect of high specific activity on the crystal structure of 238Pu nuclide thin layers was studied either.

CT-assessed large airway involvement and lung function decline in eosinophilic asthma: The association between induced sputum eosinophil differential counts and airway remodeling.

PubMed

Inoue, Hideki; Ito, Isao; Niimi, Akio; Matsumoto, Hisako; Matsuoka, Hirofumi; Jinnai, Makiko; Takeda, Tomoshi; Oguma, Tsuyoshi; Otsuka, Kojiro; Nakaji, Hitoshi; Tajiri, Tomoko; Iwata, Toshiyuki; Nagasaki, Tadao; Kanemitsu, Yoshihiro; Mishima, Michiaki

2016-11-01

Eosinophilic asthma (EA) is a distinct clinical phenotype characterized by eosinophilic airway inflammation and airway remodeling. Few studies have used computed tomography (CT) scanning to assess the association between sputum eosinophil differential counts and airway involvement. We aimed to investigate the clinical characteristics and airway involvement of EA, and to examine the correlation between induced sputum eosinophil differential counts and CT-assessed airway remodeling. We retrospectively divided 63 patients with stable asthma receiving inhaled corticosteroids into 2 groups: 26 patients with EA (sputum eosinophil >3%) and 37 patients with non-eosinophilic asthma (NEA). Clinical measurements such as spirometry, fractional exhaled nitric oxide levels (FeNO), and CT-assessed indices of airway involvement were compared between the groups. Multivariate analysis was performed to identify determinants of the percentage of wall area (WA%). The EA group had significantly longer asthma duration, lower pulmonary function, and higher FeNO than the NEA group. Also, the EA group had higher WA% and smaller airway luminal area than the NEA group. Sputum eosinophil differential counts and WA% were positively correlated. The multivariate linear regression analysis showed that the factors associated with WA% included sputum eosinophil differential counts, age, and body mass index. However, asthma duration was not associated with WA%. Our CT-assessed findings demonstrated large airway involvement in EA, and we observed a positive association between induced sputum eosinophil differential counts and WA%. The findings indicate that induced sputum eosinophil differential counts may be associated with airway remodeling in patients with stable asthma.

Microscopic Analysis and Quality Assessment of Induced Sputum From Children With Pneumonia in the PERCH Study.

PubMed

Murdoch, David R; Morpeth, Susan C; Hammitt, Laura L; Driscoll, Amanda J; Watson, Nora L; Baggett, Henry C; Brooks, W Abdullah; Deloria Knoll, Maria; Feikin, Daniel R; Kotloff, Karen L; Levine, Orin S; Madhi, Shabir A; O'Brien, Katherine L; Scott, J Anthony G; Thea, Donald M; Ahmed, Dilruba; Awori, Juliet O; DeLuca, Andrea N; Ebruke, Bernard E; Higdon, Melissa M; Jorakate, Possawat; Karron, Ruth A; Kazungu, Sidi; Kwenda, Geoffrey; Hossain, Lokman; Makprasert, Sirirat; Moore, David P; Mudau, Azwifarwi; Mwaba, John; Panchalingam, Sandra; Park, Daniel E; Prosperi, Christine; Salaudeen, Rasheed; Toure, Aliou; Zeger, Scott L; Howie, Stephen R C

2017-06-15

It is standard practice for laboratories to assess the cellular quality of expectorated sputum specimens to check that they originated from the lower respiratory tract. The presence of low numbers of squamous epithelial cells (SECs) and high numbers of polymorphonuclear (PMN) cells are regarded as indicative of a lower respiratory tract specimen. However, these quality ratings have never been evaluated for induced sputum specimens from children with suspected pneumonia. We evaluated induced sputum Gram stain smears and cultures from hospitalized children aged 1-59 months enrolled in a large study of community-acquired pneumonia. We hypothesized that a specimen representative of the lower respiratory tract will contain smaller quantities of oropharyngeal flora and be more likely to have a predominance of potential pathogens compared to a specimen containing mainly saliva. The prevalence of potential pathogens cultured from induced sputum specimens and quantity of oropharyngeal flora were compared for different quantities of SECs and PMNs. Of 3772 induced sputum specimens, 2608 (69%) had <10 SECs per low-power field (LPF) and 2350 (62%) had >25 PMNs per LPF, measures traditionally associated with specimens from the lower respiratory tract in adults. Using isolation of low quantities of oropharyngeal flora and higher prevalence of potential pathogens as markers of higher quality, <10 SECs per LPF (but not >25 PMNs per LPF) was the microscopic variable most associated with high quality of induced sputum. Quantity of SECs may be a useful quality measure of induced sputum from young children with pneumonia. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

The Most Common Detected Bacteria in Sputum of Patients with the Acute Exacerbation of COPD

PubMed Central

Cukic, Vesna

2013-01-01

Introduction: Acute exacerbation of COPD (AECOPD) may be triggered by infection with bacteria or viruses or by environmental pollutants; the cause of about one-third of exacerbations cannot be identified. Objective: To determine the most common bacteria in sputum culture of patients with AECOPD hospitalized in Intensive care unit of Clinic for pulmonary disease and TB “Podhrastovi” in the 2012. Material and methods: This is a retrospective analysis of sputum bacterial cultures of patients with AECOPD treated in the Intensive care unit of Clinic for pulmonary disease and TB “Podhrastovi” during 2012 .year. Each patient was required to give two sputum for bacterial examination. Each patient was treated with antibiotics prior to admission in Clinic “Podhrastovi”. The results of sputum bacterial culture findings are expressed in absolute number and percentage of examined patients. Results: In 2012, 75 patients with AECOPD were treated in Intensive care unit of Clinic for pulmonary disease and TB“Podhrastovi”. 44 (58.66%) of patients had normal –nonpathogenic – usual bacterial flora isolated in sputum cultures, 31 (41.34%) had a pathogen bacteria in sputum culture as follows: 7 had Streptoccocus pneumoniae, 8 had Klebsiella pneumoniae (2 with Streptococcus pneumoniae, one with Acinetobacter baumani) ,4 Escherichia colli, others are one or two cases with other bacteria. Conclusion: Bacterial airway infections play a great role in many, but not in all, of cases of AECOPD. So there is the need to do a sputum bacterial culture examination in each patient with AECOPD and with appropriate antibiotics to contribute to curing of them. PMID:24511262

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sebastien, P.; Bignon, J.; Barris, Y.I.

The counts of ferruginous bodies (pulmonary-coated mineral fibers) in sputum samples from inhabitants in each of the two agricultural villages of Karain and Tuskoey (Cappadocia, central Turkey) increased with subjects' age, whereas 94% of samples collected in the neighboring villages were free of ferruginous body. These findings are consistent with the previous hypothesis that the very high frequency of mesothelioma in Karain and Tuskoey was related to exposure to airborne mineral fibers of natural origin.

Incidence and Carrier Frequency of CFTR Gene Mutations in Pregnancies With Echogenic Bowel in Nova Scotia and Prince Edward Island.

PubMed

Miller, Michelle E; Allen, Victoria M; Brock, Jo-Ann K

2018-03-01

Fetal echogenic bowel (echogenic bowel) is associated with cystic fibrosis (CF), with a reported incidence ranging from 1% to 13%. Prenatal testing for CF in the setting of echogenic bowel can be done by screening parental or fetal samples for pathogenic CFTR variants. If only one pathogenic variant is identified, sequencing of the CFTR gene can be undertaken, to identify a second pathogenic variant not covered in the standard screening panel. Full gene sequencing, however, also introduces the potential to identify variants of uncertain significance (VUSs) that can create counselling challenges and cause parental anxiety. To provide accurate counselling for families in the study population, the incidence of CF associated with echogenic bowel and the carrier frequency of CFTR variants were investigated. All pregnancies for which CF testing was undertaken for the indication of echogenic bowel (from Nova Scotia and Prince Edward Island) were identified (January 2007-July 2017). The CFTR screening and sequencing results were reviewed, and fetal outcomes related to CF were assessed. A total of 463 pregnancies with echogenic bowel were tested. Four were confirmed to be affected with CF, giving an incidence of 0.9% in this cohort. The carrier frequency of CF among all parents in the cohort was 5.0% (1 in 20); however, when excluding parents of affected fetuses, the carrier frequency for the population was estimated at 4.1% (1 in 25). CFTR gene sequencing identified an additional VUS in two samples. The incidence of CF in pregnancies with echogenic bowel in Nova Scotia and Prince Edward Island is 0.9%, with an estimated population carrier frequency of 4.1%. These results provide the basis for improved counselling to assess the risk of CF in the pregnancy, after parental carrier screening, using Bayesian probability. Counselling regarding VUSs should be undertaken before gene sequencing. Copyright © 2017 Society of Obstetricians and Gynaecologists of Canada. Published by Elsevier Inc. All rights reserved.

The luminosity function for different morphological types in the CfA Redshift Survey

NASA Technical Reports Server (NTRS)

Marzke, Ronald O.; Geller, Margaret J.; Huchra, John P.; Corwin, Harold G., Jr.

1994-01-01

We derive the luminosity function for different morphological types in the original CfA Redshift Survey (CfA1) and in the first two slices of the CfA Redshift Survey Extension (CfA2). CfA1 is a complete sample containing 2397 galaxies distributed over 2.7 steradians with m(sub z) less than or equal 14.5. The first two complete slices of CfA2 contain 1862 galaxies distributed over 0.42 steradians with m(sub z)=15.5. The shapes of the E-S0 and spiral luminosity functions (LF) are indistinguishable. We do not confirm the steeply decreasing faint end in the E-S0 luminosity function found by Loveday et al. for an independent sample in the southern hemisphere. We demonstrate that incomplete classification in deep redshift surveys can lead to underestimates of the faint end of the elliptical luminosity function and could be partially responsible for the difference between the CfA survey and other local field surveys. The faint end of the LF for the Magellanic spirals and irregulars is very steep. The Sm-Im luminosity function is well fit by a Schechter function with M*=-18.79, alpha=-1.87, and phi*=0.6x10(exp -3) for M(sub z) less than or equal to -13. These galaxies are largely responsible for the excess at the faint end of the general CfA luminosity function. The abundance of intrinsically faint, blue galaxies nearby affects the interpretation of deep number counts. The dwarf population increases the expected counts at B=25 in a no-evolution, q(sub 0)=0.05 model by a factor of two over standard no-evolution estimates. These dwarfs change the expected median redshift in deep redshift surveys by less than 10 percent . Thus the steep Sm-Im LF may contribute to the reconciliation of deep number counts with deep redshift surveys.

Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer.

PubMed

Guttery, David S; Page, Karen; Hills, Allison; Woodley, Laura; Marchese, Stephanie D; Rghebi, Basma; Hastings, Robert K; Luo, Jinli; Pringle, J Howard; Stebbing, Justin; Coombes, R Charles; Ali, Simak; Shaw, Jacqueline A

2015-07-01

Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a "liquid biopsy." We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease. © 2015 American Association for Clinical Chemistry.

MISLEADING SPUTUM CARTONS

PubMed Central

Thomas, C. G. A.; Gibbs, F. J.

1960-01-01

Waxed paper sputum cartons obtained from the manufacturers contained very few organisms. Similar cartons which had been stored in hospital wards were often contaminated with hospital dust. This contamination was sometimes sufficiently marked to be misleading. Coliform organisms are particularly liable to cause confusion. Waxed cartons are useful for collecting sputum, but they must be properly stored. PMID:13855052

Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

2013-06-01

A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.