| 1. |
- Ruilope, LM, et al.
(author)
-
Design and Baseline Characteristics of the Finerenone in Reducing Cardiovascular Mortality and Morbidity in Diabetic Kidney Disease Trial
- 2019
-
In: American journal of nephrology. - : S. Karger AG. - 1421-9670 .- 0250-8095. ; 50:5, s. 345-356
-
Journal article (peer-reviewed)abstract
- <b><i>Background:</i></b> Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. <b><i>Patients and</i></b> <b><i>Methods:</i></b> The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate ≥25 mL/min/1.73 m<sup>2</sup> and albuminuria (urinary albumin-to-creatinine ratio ≥30 to ≤5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level α = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. <b><i>Conclusions:</i></b> FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
- Castellani, Joëlle, et al.
(author)
-
Impact of Improving Community-Based Access to Malaria Diagnosis and Treatment on Household Costs.
- 2016
-
In: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. - : Oxford University Press (OUP). - 1537-6591. ; 63:suppl 5
-
Journal article (peer-reviewed)abstract
- Community health workers (CHWs) were trained in Burkina Faso, Nigeria, and Uganda to diagnose febrile children using malaria rapid diagnostic tests, and treat positive malaria cases with artemisinin-based combination therapy (ACT) and those who could not take oral medicines with rectal artesunate. We quantified the impact of this intervention on private household costs for childhood febrile illness.Households with recent febrile illness in a young child in previous 2 weeks were selected randomly before and during the intervention and data obtained on household costs for the illness episode. Household costs included consultation fees, registration costs, user fees, diagnosis, bed, drugs, food, and transport costs. Private household costs per episode before and during the intervention were compared. The intervention's impact on household costs per episode was calculated and projected to districtwide impacts on household costs.Use of CHWs increased from 35% of illness episodes before the intervention to 50% during the intervention (P < .0001), and total household costs per episode decreased significantly in each country: from US Dollars (USD) $4.36 to USD $1.54 in Burkina Faso, from USD $3.90 to USD $2.04 in Nigeria, and from USD $4.46 to USD $1.42 in Uganda (all P < .0001). There was no difference in the time used by the child's caregiver to care for a sick child (59% before intervention vs 51% during intervention spent ≤2 days). Using the most recent population figures for each study district, we estimate that the intervention could save households a total of USD $29 965, USD $254 268, and USD $303 467, respectively, in the study districts in Burkina Faso, Nigeria, and Uganda.Improving access to malaria diagnostics and treatments in malaria-endemic areas substantially reduces private household costs. The key challenge is to develop and strengthen community human resources to deliver the intervention, and ensure adequate supplies of commodities and supervision. We demonstrate feasibility and benefit to populations living in difficult circumstances.ISRCTN13858170.
|
|
| 7. |
|
|
| 8. |
- Castellani, J., et al.
(author)
-
Out-of-Pocket Costs and Other Determinants of Access to Healthcare for Children with Febrile Illnesses: A Case-Control Study in Rural Tanzania
- 2015
-
In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 10:4
-
Journal article (peer-reviewed)abstract
- Objectives To study private costs and other determinants of access to healthcare for childhood fevers in rural Tanzania. A case-control study was conducted in Tanzania to establish factors that determine access to a health facility in acute febrile illnesses in children less than 5 years of age. Carers of eligible children were interviewed in the community; cases were represented by patients who went to a facility and controls by those who did not. A Household Wealth Index was estimated using principal components analysis. A multivariable logistic regression analysis was performed to understand the factors which influenced attendance of healthcare facility including severity of the illness and household wealth/socio-demographic indicators. To complement the data on costs from community interviews, a hospital-based study obtained details of private expenditures for hospitalised children under the age of 5. Severe febrile illness is strongly associated with health facility attendance (OR: 35.76, 95% CI: 3.68-347.43, p = 0.002 compared with less severe febrile illness). Overall, the private costs of an illness for patients who went to a hospital were six times larger than private costs of controls ($5.68 vs. $0.90, p<0.0001). Household wealth was not significantly correlated with total costs incurred. The separate hospital based cost study indicated that private costs were three times greater for admissions at the mission versus public hospital: $13.68 mission vs. $4.47 public hospital (difference $9.21 (95% CI: 7.89 - 10.52), p<0.0001). In both locations, approximately 50% of the cost was determined by the duration of admission, with each day in hospital increasing private costs by about 12% (95% CI: 5% - 21%). The more severely ill a child, the higher the probability of attending hospital. We did not find association between household wealth and attending a health facility; nor was there an association between household wealth and private cost.
|
|
| 9. |
- Gutierrez-Suarez, R., et al.
(author)
-
Health-related quality of life of patients with juvenile idiopathic arthritis coming from 3 different geographic areas. The PRINTO multinational quality of life cohort study
- 2007
-
In: Rheumatology (Oxford). - : Oxford University Press (OUP). - 1462-0324 .- 1462-0332. ; 46:2, s. 314-320
-
Journal article (peer-reviewed)abstract
- OBJECTIVES: To compare health-related quality of life (HRQL) and to identify clinical determinants for poor HRQL of patients with juvenile idiopathic arthritis (JIA) coming from three geographic areas.METHODS: The HRQL was assessed through the Child Health Questionnaire (CHQ). A total of 30 countries were included grouped in three geographic areas: 16 countries in Western Europe; 10 in Eastern Europe; and four in Latin America. Potential determinants of poor HRQL included demographic data, physician's and parent's global assessments, measures of joint inflammation, disability as measured by Childhood Health Assessment Questionnaire (CHAQ) and erythrocyte sedimentation rate. Poor HRQL was defined as a CHQ physical summary score (PhS) or psychosocial summary score (PsS) <2 S.D. from that of healthy children.RESULTS: A total of 3167 patients with JIA, younger than 18 yrs, were included in this study. The most affected health concepts (<2 S.D. from healthy children) that differentiate the three geographic areas include physical functioning, bodily pain/discomfort, global health, general health perception, change in health with respect to the previous year, self-esteem and family cohesion. Determinants for poor HRQL were similar across geographic areas with physical well-being mostly affected by the level of disability while the psychosocial well-being by the intensity of pain.CONCLUSION: We found that patients with JIA have a significant impairment of their HRQL compared with healthy peers, particularly in the physical domain. Disability and pain are the most important determinants of physical and psychosocial well-being irrespective of the geographic area of origin.
|
|
| 10. |
- Mihaylova, Sashka A, et al.
(author)
-
Analyses of clinically-relevant isolates of Stenotrophomonas spp.
- 2007
-
In: Proceedings of the 26th Annual General Meeting of the European Culture Collections’ Organization; A0264.
-
Conference paper (peer-reviewed)abstract
- Objectives: The identification of clinical isolates of Stenotrophomonas spp. to the species level, using a polyphasic approach of phenotypic and genotypic methods, and the determination and comparison of the in vitro responses to various classes of antimicrobial agents by these isolates. Previous studies have suggested a marked diversity among clinical, as well as environmental, isolates of Stenotrophomonas spp. and they represent a significant threat within hospital settings, particularly for patients in ICUs and other immunocompromised conditions. Isolates characterised as putative Stenotrophomonas spp. from Pleven University Hospital were analysed in detail. The predominant source of the isolates was respiratory tract and two thirds of patients were admitted to ICUs. Isolates were examined, using the VITEK 2 compact system, CCUG phenotypic characterisation panels and sequencing and analyses of the genes for 16S rRNA and gyrB. The in vitro responses of isolates to 10 antibiotics (azlocillin, piperacillin, ceftazidime, imipenem, meropenem, ampicillin-sulbactam, piperacillin-tazobactam, gentamicin, amikacin and ciprofloxacin) were evaluated, using the disc diffusion method, according to guidelines of the Clinical and Laboratory Standards Institute. The commercial VITEK 2 system defined all isolates as S. maltophilia, with probabilities of identifications varying from 92 to 99%, and “good”, “very good” or “excellent” confidence. Results from conventional phenotypic testing confirmed identifications for isolates as S. maltophilia or S. rhizophila. DNA sequencing and analyses further defined the species affiliations of isolates of the genus Stenotrophomonas. Previous reports have indicated that Stenotrophomonas spp. exhibit resistance to a broad range of antibiotics. More than 90% of examined isolates were observed to be resistant to imipenem, meropenem and ampicillin-sulbactam, more than 80% were resistant to azlocillin, piperacillin and piperacillin-tazobactam. The only beta-lactam antibiotic with an indication of better activity was ceftazidime. Among the ten antimicrobial agents tested, ciprofloxacin demonstrated the highest activity, as evaluated by diameter zone of growth inhibition. Conclusions: The VITEK 2 compact system identified all isolates to the genus level. However, conventional phenotypic testing may be more effective for species differentiation. Analysis of DNA gene sequencing allows further precise taxonomic identification. Ciprofloxacin could be a drug of choice for therapy of infections caused by Stenotrophomonas spp.
|
|
| 11. |
- Mihaylova, Sashka A, et al.
(author)
-
Emergence of clinical strains of Stenotrophomonas maltophilia resistant to trimethoprim/sulfamethoxazole in a Bulgarian university hospital
- 2008
-
In: Journal of Biomedical and Clinical Research. - 1313-9053. ; 1, s. 18-25
-
Journal article (peer-reviewed)abstract
- Clinical strains of Stenotrophomonas maltophilia, identified by a commercial system as resistant to trimethoprim/sulfamethoxazole (TMP/SMX), were analysed in detail, to confirm their taxonomic positions, to determine their susceptibilities to various classes of antibiotics and to assess this information with respect to the epidemiological relevance. The majority of strains were isolated from respiratory and wound specimens from patients admitted to intensive care units. Multi-locus sequence analyses (MLSA) of 16S ribosomal RNA (rRNA) and gyrase subunit B (gyrB) genes were applied for genotypic-based characterisation. The minimal inhibitory concentrations (MICs) of ten antimicrobial agents were determined, using the E-test method. The MIC values of TMP/SMX for the clinical isolates of S. maltophilia were greater than 32 mg/L, which confirmed their preliminary assessment as resistant. Minocycline, levofloxacin and ciprofloxacin exhibited the lowest MICs. All strains were observed to be susceptible to minocycline and levofloxacin. The emergence of clinical strains of S. maltophilia resistant to TMP/SMX is increasingly problematic as this antimicrobial agent is accepted as the “drug of choice” for treating infections caused by this bacterium. However, minocycline and levofloxacin demonstrated excellent in vitro activities and could be considered as alternative options to counter TMP/SMX-resistant strains of S. maltophilia.
|
|
| 12. |
- Mihaylova, Sashka A, et al.
(author)
-
In vitro activity of antimicrobial agents against extended-spectrum-beta-lactamase-producing enteric bacteria isolated from urine specimens
- 2009
-
In: FEMS 2009 - 3rd Congress of European Microbiologists.
-
Conference paper (other academic/artistic)abstract
- Objectives: To apply detailed phenotypic and genotypic analyses on extended-spectrum-beta-lactamase (ESBL)-producing clinical isolates. To determine in vitro activities of 14 antimicrobial agents and access these data with respect to treatment choice. Methods: One hundred strains were collected from urine samples of patients from Pleven University Hospital, Bulgaria, from January to December 2008. Patient ages: one month to 85 years. Two thirds were male. At the time of sampling, 50 patients were in Urology department, 23 were in Nephrology department, 8 were in Pediatric department, and 19 were in other departments. Double disc and Combination disc tests were applied for ESBL confirmation. ESBL-types were determined, using a multiplex PCR assay for CTX, TEX, SHV and OXA types. In vitro activities of fosfomycin, imipenem, meropenem, ertapenem, amoxicillin/clavulanic acid, piperacillin/tazobactam, gentamicin, amikacin, tigecycline, colistin, nalidixic acid, nitrofurantoin, ciprofloxacin and levofloxacin were determined by the disk diffusion method, according to guidelines of the CLSI. Results: All strains were confirmed to be members of the Enterobacteriaceae and to be ESBL producers. All strains were susceptible to the three carbapenems tested. Eighty-eight percent of the strains were susceptible to tigecycline, 74% were susceptible to amikacin, 64% were susceptible to piperacillin/tazobactam and 63% were susceptible to nitrofurantoin. More than 70% of the strains tested were resistant or intermediately susceptible to amoxicillin/clavulanic acid, gentamicin, nalidixic acid, ciprofloxacin and levofloxacin. Conclusions: Correlation was found between phenotypic and genotypic methods for detection of ESBL-producing bacteria. Carbapenems demonstrated excellent in vitro activity against strains of ESBL-positive enteric bacteria isolated in a Bulgarian university hospital. Tigecycline, amikacin, piperacillin/tazobactam, nitrofurantoin should also be considered as treatment options.
|
|
| 13. |
- Mihaylova, S., et al.
(author)
-
Susceptibility of environmental strains of Rhizobium radiobacter to antimicrobial agents
- 2014
-
In: World Applied Sciences Journal. - : International Digital Organization for Scientific Information. - 1818-4952. ; 31:5, s. 859-862
-
Journal article (peer-reviewed)abstract
- The objective of the present study was to evaluate the in vitro activities of antimicrobial agents against Rhizobium radiobacter strains, in order to identify the most effective antibiotics for the elimination and control of this bacterium from different biological systems. Carbenicillin, imipenem, meropenem, gentamicin, amikacin, polymyxin B, ciprofloxacin and levofloxacin were determined to be applicable for transformation of the plants with these bacteria. Carbenicillin, carbapenems and fluoroquinolones would be suggested for treatment of human infections. © IDOSI Publications, 2014.
|
|
| 14. |
- Moore, Edward R.B. 1954, et al.
(author)
-
Applications of MALDI-TOF and SARAMIS analyses for the identification of closely related clinically-relevant bacterial species
- 2009
-
In: Proceedings of the 12th Conference in Genomics and Proteomics of Human Pathogens: Target molecules and biomarkers in the characterisation of microbes in disease and the environment, June 25-26, London, UK.
-
Conference paper (peer-reviewed)abstract
- Identification of prokaryotes in the complexity of microbial diversity is increasingly problematic for clinical diagnoses. Among the most difficult problems for clinical diagnostics is the identification of bacteria within “complexes” of closely related species, comprising pathogenic and non-pathogenic species with limited differentiating characteristics. However, these species complexes are composed of micro-organisms with different virulence potentials and it is essential to be able to obtain and confirm reliable identifications. A “polyphasic” multi-locus sequence analysis (MLSA) strategy may be applied for the typing and identification (species- and sub-species-levels) of bacteria, including “first-phase” comparisons of partial 16S rRNA gene sequences and subsequent “second-phase” analyses of one or more conserved house-keeping genes, for identification to the species level. These genotypic data are being used to correlate with the data obtained by MALDI-TOF (matrix-assisted laser desorption-ionisation time-of-flight) IC-MS (intact-cell mass-spectrometry) and analysed with the SARAMIS (Spectral Archive And Microbial Identification System) software and database package. The high reproducibility, rapid speed and low cost of MALDI-TOF IC-MS analyses makes the methodology ideal for processing large numbers of microbiological samples sent to the clinical lab for identification. The question of the resolution able to be obtained, using MALDI-TOF IC-MS was addressed by focusing analyses on bacteria comprising clinically-relevant species-complexes. The SARAMIS analysis of the MALDI-TOF IC-MS data was observed to be able to compare spectra from bacteria, yeast and fungi with reference spectra maintained in a database, identify the most closely related bacterial species with statistically-relevant confidence values and type strains to sub-species levels. The analyses are applicable to nearly all micro-organisms with high reproducibility.
|
|
| 15. |
|
|
| 16. |
- Moore, Edward R.B. 1954, et al.
(author)
-
Microbial systematics and taxonomy: relevance for a microbial commons.
- 2010
-
In: Research in microbiology. - : Elsevier BV. - 1769-7123 .- 0923-2508. ; 161:6, s. 430-8
-
Research review (peer-reviewed)abstract
- The issues of microbial taxonomy and potential interactions with a microbial commons are discussed, with emphasis on three components: characterization; classification; and nomenclature. The current state of technology and the spectrum of methods that are used for phenotypic and genotypic characterization of prokaryotes, classification at different taxonomic levels and points of prokaryote nomenclature are reviewed. While all taxonomic ranks comprise a cohesive systematic framework for microorganisms, the prokaryotic genus and species provide the "working unit" of taxonomy. Since 2004, the number of validly published genera and species has increased by approximately 50%. Extensive development of technology will continue to enable ever higher resolution characterization and more refined classification of microorganisms. Characterization and classification at the species level may be most relevant for bacterial taxonomy, although reproducible differentiation at the strain level will probably prove to be more relevant for a microbial commons. A dynamic microbial taxonomy, albeit with well-founded and stable guidelines for defining microorganisms, provides an efficient organizational system for dealing with the enormous spectrum of microbial diversity.
|
|
| 17. |
- Moore, Edward R.B. 1954, et al.
(author)
-
Polyphasic phenotypic and genotypic analyses for diagnosing closely related clinically-relevant bacteria
- 2007
-
In: Proceedings of the XXIX Congreso Chileno de Microbología, December 3-5, Viña del Mar, Chile. ; , s. 44-45
-
Conference paper (peer-reviewed)abstract
- The majority of known pathogenic bacteria have been recognised for more than a century. However, reliable identification of specific pathogens within the increasing complexity of bacterial diversity is becoming more problematic for clinical diagnoses. DNA sequencing and genotyping of bacteria has helped enable recognition of the extensive diversity of microorganisms in the environment and the same approaches are more recently being adopted in clinical microbiology. Genotypic methods are especially suited to the analyses of the “difficult-to-cultivate” organisms, as well as bacteria that pose significant health risks during cultivation. Traditional phenotyping, as well as genotyping by 16S rRNA gene sequence analysis, in many cases are able to provide only good estimations of identifications, although they may be able to provide information on what an isolate is not. In some cases of clinical diagnoses, such information may be useful and adequate. However, such analyses are often not able to provide definitive identifications. Among the most difficult problem for diagnoses in clinical cases, is the identification of organisms within “complexes” of closely related species, e.g., the Burkholderia cepacia “complex”, the Mycobacterium tuberculosis “complex”, the Streptococcus mitis “complex”, etc. These, so called “complexes” are comprised of closely related pathogenic and non-pathogenic species or genomovars, with limited phenotypic and genotypic differentiating characteristics, e.g., the 16S rRNA gene sequence differences among such organisms is less than 1.0%. Because these species “complexes” are comprised of organisms with different pathogenic and virulence potential and different treatment regimen, it is essential to be able to provide reliable identifications to the clinicians. Genotyping of bacteria provides the means for detailed, high-resolution differentiation and identification of organisms, as well as epidemiological monitoring. For identification of clinical isolates, a “multi-locus sequence analysis (MLSA)”, is used, including “first-phasic” comparisons of partial 16S rRNA gene sequences, for identification to the sub-genus level, and subsequent analyses of one or more “house-keeping” genes, for identification to the species level. However, not all house-keeping genes are equally useful for all taxa. Thus, the key to effective identifications, with respect to the speed and cost of analyses, and the resolution and reproducibility of identifications, depends upon the selection of the most applicable house-keeping genes, i.e., those with adequately high levels of resolution among the most closely related species. Protocols and results for species-resolving MLSA, using “house-keeping” genes enable effective differentiation of clinically-relevant closely related species of respective genera of Burkholderia, Mycobacterium, Streptococcus and Staphylococcus, as well as Pseudomonas and Stenotrophomonas. Furthermore, Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass-spectrometry (MALDI-TOF MS), in combination with Spectral Archive And Microbial Identification System (SARAMIS) software analyses, were able to further define species-level differentiation among the most closely-related members of species “complexes”. MALDI-TOF MS, generating cell protein profiles, may exhibit even higher levels of resolution than the most discriminating genotypic analyses for species, as well as strain, identifications. These phenotypic data, as well as traditional phenotypic data, and genotypic MLSA data are correlated in the polyphasic assessment and definition of selected species “complexes”.
|
|
| 18. |
- Moore, Edward R.B. 1954, et al.
(author)
-
The family cardiobacteriaceae
- 2014
-
In: The Prokaryotes: Gammaproteobacteria. - Berlin, Heidelberg : Springer. - 9783642389221 ; 9783642389221, s. 135-146
-
Book chapter (peer-reviewed)abstract
- Cardiobacteriaceae, within the order, Cardiobacteriales, in the class, Gammaproteobacteria, was described as a novel family, based upon the 16S rRNA sequence-based relationships of Cardiobacterium hominis, Dichelobacter nodosus, and Suttonella indologenes, representing distinct lineages within a common phylogenetic cluster. The two species of Cardiobacterium, one species of Dichelobacter, and two species of Suttonella are Gram-negative, rod-shaped, fastidious, facultatively anaerobic, strictly anaerobic or microaerophilic chemoorganotrophs, manifesting pathogenicity in humans and animals. Strains of the species are isolated from human and animal samples. C. hominis, C. valvarum, and S. indologenes are typically recovered from the blood cultures of patients with endocarditis. D. nodosus is a causative agent for foot-rot disease in cloven-hoofed animals, and S. ornithocola is implicated in pulmonary necrosis in birds of the tit family. Rarely are bacteria of the Cardiobacteriaceae detected in nonclinical samples. © 2014 Springer-Verlag Berlin Heidelberg. All rights are reserved.
|
|
| 19. |
- Ruperto, N., et al.
(author)
-
PRINTO/PRES international website for families of children with rheumatic diseases: www.pediatric-rheumatology.printo.it
- 2005
-
In: Ann Rheum Dis. - : BMJ. - 0003-4967. ; 64:7, s. 1101-6
-
Journal article (peer-reviewed)abstract
- OBJECTIVE: To prepare a website for families and health professionals containing up to date information about paediatric rheumatic diseases (PRD). METHODS: Firstly, paediatric rheumatology centres and family self help associations were surveyed to characterise current clinical practice of physicians providing care for children with PRD, research activities, and training facilities of each centre. Secondly, international consensus was reached on the content of the website. Finally, the website was developed and the texts translated. RESULTS: The web page contains three main sections: (a) description for families of the characteristics of 15 PRD; (b) list of paediatric rheumatology centres; (c) contact information for family self help associations. A version for 45 countries in 52 languages (with another three in progress) is now available on the web. 291 surveys from 171 centres and 102 family associations were received from 42 countries. The median proportion of time spent in paediatric practice in the centres examined was 100%, with 70% of this time dedicated to paediatric rheumatology. 90% of the centres were willing to perform clinical trials in the future. CONCLUSIONS: The PRINTO/PRES website provides a well defined and competent set of information about PRD, with appropriate multiple translated versions and easy web navigational direction.
|
|
| 20. |
|
|
| 21. |
- Svensson, Liselott A, et al.
(author)
-
New genotypic and phenotypic analyses of clinically-relevant Gram-negative, non-fermenting bacteria: MALDI-TOF MS as a rapid, high-resolution method for identifying and typing microorganisms
- 2010
-
In: 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Vienna, Austria.
-
Conference paper (other academic/artistic)abstract
- Objectives. Identification of Gram-negative, non-fermenting bacilli, using phenotypic characterization is problematic. Many of the species of this group are frequent nosocomial infectious agents and are ubiquitous in the environment. The aims of this study were to assess the resolving capacities of “house-keeping” gene sequences, including 16S rRNA, atpD, gyrB, recA, rpoB and rpoD, and to compare a multi-locus sequence analysis (MLSA) with matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry analyses for identifying and typing strains of Achromobacter, Bordetella, Burkholderia, Pseudomonas, and Stenotrophomonas species. Methods. Genotypic analyses. Type strains of the focus genera and species-complexes, other well-characterised reference strains and selected clinically-relevant strains representing a range of phenotypic and genotypic similarities were included in this study. Partial genes, 16S rRNA, atpD, recA, gyrB and rpoD recA were amplified by PCR and sequenced. DNA-DNA hybridisation analyses were carried out on selected strains for confirmation of species designations. MALDI-TOF analysis. Bacterial biomass were prepared from cultures on agar medium and analysed by MALDI-TOF MS, in the positive mode, using the SARAMIS software for analysis (1) Results. MLSA, using the respective house keeping genes were able to differentiate and identify the most closely related species of the analysed taxa and cluster analyses showed similarities of branching order between species that correlated well between different genes. However, different genes were not equally effective in differentiating species of the different genera. The MALDI-TOF analyses were effective in differentiating the most closely related species of the respective genera. Good correlation was observed between the results of MALDI-TOF MS and MLSA data. Conclusion. In most cases, clinically-relevant isolates and strains of Gram negative, non-fermenting bacilli exhibited good agreement between the methods of this study. In some cases, strains previously defined as given species were observed to be genotypically more similar to other species, as well as some strains with highly aberrant phenotypes were almost genotypically identical to the type strain. MALDI-TOF identification was very well correlated to the MLSA results, and is a much less expensive and effectively able to reduce identification times by 24-48 hours. (1) Vanlaere E et al. J. Microbiol. Meth. 75: 279-286 (2008).
|
|
| 22. |
- Svensson, Liselott A, et al.
(author)
-
Stenotrophomonas species differentiation by MLSA of gyrB and rpoD and identification of isolates from clinical and environmental samples
- 2009
-
In: FEMS 2009 - 3rd Congress of European Microbiologists.
-
Conference paper (other academic/artistic)abstract
- Background: Stenotrophomonas species are commonly observed in clinical and environmental samples. S. maltophilia is associated with respiratory infections, is recognized as the third most common nosocomial Gram-negative, non-fermenting bacterium and is isolated from a range of environmental ecosystems, often exhibiting biodegradation and other biotechnological potential. Identification of Stenotrophomonas spp. is famously problematic, due to lack of phenotypic markers, as well as limited 16S rRNA gene sequence differentiation. Objectives: The aim of this study was to determine the applicability of “house-keeping” genes gyrB and rpoD as biomarkers for species-level differentiation within Stenotrophomonas. Methods: Selected regions from gyrB and rpoD of Stenotrophomonas spp., including all type strains, were amplified by PCR and sequenced, for comparative analyses. Results: Comparative sequence analyses of targeted regions of gyrB and rpoD enabled effective resolution of all species in Stenotrophomonas. Dissimilarities between the sequences of the type strains of S. maltophilia and other Stenotrophomonas spp. ranged from 10.2 – 14.7 % (gyrB) and 13.0 – 21.7 % (rpoD). Inter-species sequence dissimilarities of gyrB and rpoD ranged from 7.2 % --18.0 % and from 5.5 % - 22.8%, respectively. Compared with Stenotrophomonas spp. 16S rRNA gene sequence dissimilarities, (0.9 – 3.1 %), both house keeping genes exhibit better capacity for reliably differentiating Stenotrophomonas species. Clinical isolates identified as S. maltophilia were analysed by gyrB and rpoD sequence analyses. These data elucidated high levels of sequence dissimilarities among isolates, bringing into question the identifications determined by traditional methods, as well as the resolution of the taxonomy of Stenotrophomonas. These questions are being addressed in more detail. Conclusions: Analyses of house-keeping genes gyrB and rpoD exhibited effective species-level differentiation for Stenotrophomonas, which can be applied, in combination with 16S rRNA gene sequence analyses to provide an MLSA strategy for genotypic analyses for reliable identifications of isolates from clinical and environmental samples.
|
|
| 23. |
- Svensson-Stadler, Liselott, 1971, et al.
(author)
-
Stenotrophomonas interspecies differentiation and identification by gyrB sequence analysis.
- 2012
-
In: FEMS microbiology letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 327:1, s. 15-24
-
Journal article (peer-reviewed)abstract
- Stenotrophomonas species are found commonly in environmental and clinical samples; Stenotrophomonas maltophilia is an important opportunistic pathogen of humans. Traditional phenotyping protocols, as well as genotyping by 16S rRNA gene sequence analysis, do not reliably distinguish the species of Stenotrophomonas. Sequence analyses of two targeted PCR-amplified regions of the gyrB gene, which encodes the β-subunit of DNA gyrase, enabled resolution and identification of these species. Most type strains of the different species of Stenotrophomonas exhibited more than 7% dissimilarity in the gyrB gene sequences. Among these, strains identified as the same species exhibited sequence dissimilarities up to 4.6% and 5.9% for the two regions, respectively. Strains identified as S. maltophilia, with 16S rRNA gene sequence similarities > 99.0%, were grouped within a 'S. maltophilia complex'; these organisms exhibited gyrB similarities as low as 93%. Many of these strains possessed genomic DNA similarities with the type strain of S. maltophilia CCUG 5866(T) below 70%. These data, including gyrB sequence comparisons, indicate that strains identified as S. maltophilia may comprise distinct, new species.
|
|
| 24. |
- Wang, Ellen, et al.
(author)
-
Automated functional characterization of radiolabeled antibodies : a time-resolved approach
- 2014
-
In: Nuclear medicine communications. - 0143-3636 .- 1473-5628. ; 35:7, s. 767-776
-
Journal article (peer-reviewed)abstract
- Background The number of radiolabeled monoclonal antibodies (mAbs) used for medical imaging and cancer therapy is increasing. The required chemical modification for attaching a radioactive label and all associated treatment may lead to a damaged mAb subpopulation. This paper describes a novel method, concentration through kinetics (CTK), for rapid assessment of the concentration of immunoreactive mAb and the specific radioactivity, based on monitoring binding kinetics. Methods The interaction of radiolabeled mAb with either the antigen or a general mAb binder such as Protein A was monitored in real time using the instrument LigandTracer. As the curvature of the binding trace has a distinct shape based on the interaction kinetics and concentration of the functional mAb, the immunoreactive mAb concentration could be calculated through reverse kinetic fitting of the binding curves, using software developed for this project. The specific activity, describing the degree of radioactive labeling, was determined through the use of calibrated signal intensities. Results The performance of the CTK assay was evaluated on the basis of various mAb-based interaction systems and assay formats, and it was shown that the assay can provide accurate and repeatable results for immunoreactive concentration and specific activity, with both accuracy and relative SD values below 15%. Conclusion By applying reverse kinetics on real-time binding traces it is possible to estimate the functional concentration and specific activity of radiolabeled mAb. The CTK assay may in the future be included as a complement to current quality assessment methods of radiolabeled mAbs.
|
|
