| 1. |
- Brolund, Alma, et al.
(författare)
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Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae
- 2010
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 82:3, s. 229-233
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBLM) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBLM are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla(AmpC) detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes. Material and methods: Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmo University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla(AmpC) real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing. Results and discussion: The real-time PCR assay was able to detect and sub-classify all acquired bla(AmpC) genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla(AmpC) real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated. (C) 2010 Elsevier B.V. All rights reserved.
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| 2. |
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| 3. |
- Bunikis, Ignas, et al.
(författare)
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Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi.
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 86:2, s. 243-7
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Tidskriftsartikel (refereegranskat)abstract
- Borrelia burgdorferi has an unusual genomic structure containing 21 plasmids. These plasmids carry genes that are essential for infectivity and survival of the spirochetes in vivo. Several plasmids are lost during cultivation in vitro, which might lead to a heterogeneous population after multiple passages and loss of infectivity in laboratory animals. Herein, we present a simple and inexpensive multiplex PCR method that detects the complete plasmid profile of B. burgdorferi B31 in just two PCR tubes.
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| 4. |
- Gantelius, Jesper, et al.
(författare)
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A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum
- 2010
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 82:1, s. 11-18
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Tidskriftsartikel (refereegranskat)abstract
- Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC = 97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.
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| 5. |
- Gantner, S, et al.
(författare)
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Novel primers for 16S rRNA-based archaeal community analyses in environmental samples
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 84:1, s. 12-18
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Tidskriftsartikel (refereegranskat)abstract
- Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (<1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.
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| 6. |
- Gorokhova, Elena, et al.
(författare)
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A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy
- 2012
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 89:3, s. 216-221
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Tidskriftsartikel (refereegranskat)abstract
- Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by similar to 25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in -80 degrees C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies.
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| 7. |
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| 8. |
- Hellman, Maria, et al.
(författare)
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Survey of bromodeoxyuridine uptake among environmental bacteria and variation in uptake rates in a taxonomically diverse set of bacterial isolates
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 86, s. 376-378
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Tidskriftsartikel (refereegranskat)abstract
- Incorporation of 5-Bromo-2'-Deoxyuridine (BrdU) into DNA can be used to target replicating bacteria in the environment, but differential uptake capacity is a potential bias. Among 23 bacterial isolates commonly found in soils, most took up BrdU, but at up to 10-fold different cell-specific rates. Combined with results from an in silico analysis of 1000 BrdU-labeled 16S rRNA gene sequences, our results demonstrate a BrdU uptake bias with no apparent relationship between taxa affiliation and ability to incorporate BrdU. (C) 2011 Elsevier B.V. All rights reserved.
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| 9. |
- Kalle Kossio, Elena
(författare)
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External and semi-internal controls for PCR amplification of homologous sequences in mixed templates
- 2013
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 95, s. 285-294
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Tidskriftsartikel (refereegranskat)abstract
- In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control: therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. (C) 2013 Elsevier B.V. All rights reserved.
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| 10. |
- Larsson, Marie C, et al.
(författare)
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A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability
- 2014
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 106, s. 146-150
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Tidskriftsartikel (refereegranskat)abstract
- The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5 days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4 mg/L, 8 mg/L and 2 mg/L, respectively) compared to extracellularly (MIC90: 0.5 mg/L for AMI and EMB; 0.25 mg/L for LEV). The reverse was the case for INH (IC: 0.064 mg/L vs EC: 0.25 mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs. (C) 2014 Elsevier B.V. All rights reserved.
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| 11. |
- Lu, Xuedong, et al.
(författare)
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A rapid two-step algorithm detects and identifies clinical macrolide and beta-lactam antibiotic resistance in clinical bacterial isolates
- 2014
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 102, s. 26-31
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Aiming to identify macrolide and beta-lactam resistance in clinical bacterial isolates rapidly and accurately, a two-step algorithm was developed based on detection of eight antibiotic resistance genes. Methods: Targeting at genes linked to bacterial macrolide (msrA, ermA, ermB, and ermC) and beta-lactam (bla(TEM), bla(SHV), bla(CTX-N-1), bin(CTX-M-9)) antibiotic resistances, this method includes a multiplex real-time PCR, a melting temperature profile analysis as well as a liquid bead microarray assay. Liquid bead microarray assay is applied only when indistinguishable T-m profile is observed. Results: The clinical validity of this method was assessed on clinical bacterial isolates. Among the total 580 isolates that were determined by our diagnostic method, 75% of them were identified by the multiplex real-time PCR with melting temperature analysis alone, while the remaining 25% required both multiplex real-time PCR with melting temperature analysis and liquid bead microarray assay for identification. Compared with the traditional phenotypic antibiotic susceptibility test, an overall agreement of 81.2% (kappa = 0.614, 95% Cl = 0550-0.679) was observed, with a sensitivity and specificity of 87.7% and 73% respectively. Besides, the average test turnaround time is 3.9 h, which is much shorter in comparison with more than 24 h for the traditional phenotypic tests. Conclusions: Having the advantages of the shorter operating time and comparable high sensitivity and specificity with the traditional phenotypic test, our two-step algorithm provides an efficient tool for rapid determination of macrolide and beta-lactam antibiotic resistances in clinical bacterial isolates.
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| 12. |
- Løvdal, Trond, et al.
(författare)
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Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 85:2, s. 164-169
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Tidskriftsartikel (refereegranskat)abstract
- The combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) significantly overestimated the fraction of viable Listeria innocua as compared to plate counts and confocal fluorescence microscopy. Our data imply that PMA-qPCR must be used with caution as an analytical tool for the differentiation between viable and dead bacteria.
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| 13. |
- Marcinowska, Renata, et al.
(författare)
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Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier. - 0167-7012 .- 1872-8359. ; 87:1, s. 24-31
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Tidskriftsartikel (refereegranskat)abstract
- Metabolomics, or metabolite profiling, is an approach that is increasingly used to study the metabolism of diverse organisms, elucidate biological processes and/or find characteristic biomarkers of physiological states. Here, we describe the optimization of a method for global metabolomic analysis of bacterial cultures, with the following steps. Cells are grown to log-phase, starting from an overnight culture and bacterial concentrations are monitored by measuring the optical density of the cultures at 600nm. At an appropriate density they are harvested by centrifugation, washed three times with NaCl solution and metabolites are extracted using methanol and a bead-mill. Dried extracts are methoxymated and derivatized with methyltrimethylsilyltrifluoroacetamide (MSTFA) then analyzed using gas chromatography coupled to time-of-flight mass spectrometry (GC-MS/TOF). Finally, patterns in the acquired data are examined by multivariate data modeling. This method enabled us to obtain reproducible metabolite profiles of Yersinia pseudotuberculosis, with about 25% compound identification, based on comparison with entries in available GC-MS libraries. To assess the potential utility of the method for comparative analysis of other bacterial species we analyzed cultures of Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and methicillin-sensitive Staphylococcus aureus (MSSA). Multivariate analysis of the acquired data showed that it was possible to differentiate the species according to their metabolic profiles. Our results show that the presented procedure can be used for metabolomic analysis of a wide range of bacterial species of clinical interest.
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| 14. |
- Markowicz, Pawel, et al.
(författare)
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The surface emissions trap: A new approach in indoor air purification.
- 2012
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 91:2, s. 290-294
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Tidskriftsartikel (refereegranskat)abstract
- A new device for stopping or reducing potentially irritating or harmful emissions from surfaces indoors is described. The device is a surface emissions trap prototype and consists of an adsorbent sheet with a semipermeable barrier surrounded by two thin nonwoven layers. The trap may be applied directly at the source of the emissions e.g. at moisture-affected floors and walls, surfaces contaminated by chemical spills etc. This results in an immediate stop or reduction of the emitting pollutants. The trap has a very low water vapor resistance thus allowing drying of wet surfaces. In laboratory experiments typically 98% reduction of air concentrations of volatile organic compounds and a virtually total reduction of mold particle-associated mycotoxins was observed. The surface emissions trap may represent a convenient and efficient way of restoring indoor environments polluted by microbial and other moisture-associated emissions.
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| 15. |
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| 16. |
- Ryberg, Anna, et al.
(författare)
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Comparison of (GTG)(5)-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates
- 2011
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 84:2, s. 183-188
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Tidskriftsartikel (refereegranskat)abstract
- Molecular typing of Klebsiella species has become important for monitoring dissemination of beta-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)(5)- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19K pneumoniae (some with an ESBL-phenotype). 35 K. oxytoca isolates, five K pneumoniae, two K oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)(5) and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)(5) and ITS-PCR analysis revealed that K pneumoniae and K oxytoca isolates. reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)(5) and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates. (C) 2010 Elsevier B.V. All rights reserved.
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| 17. |
- Sjöberg, Fei, et al.
(författare)
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Comparison between terminal-restriction fragment length polymorphism (T-RFLP) and quantitative culture for analysis of infants' gut microbiota
- 2013
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 94:1, s. 37-46
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Tidskriftsartikel (refereegranskat)abstract
- The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism (T-RFLP). A database was constructed for direct identification of T-RFLP peaks by analysis of pure-culture bacteria and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at >10(6) CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts. In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be identified using the database. T-RFLP readily identified bacteria to the genus level and also provided some sub-genus discrimination. Both T-RFLP and culture identified Bifidobacterium, Clostridium and Bacteroides spp. among the most common colonizers of the infantile microbiota throughout the first year of life. T-RFLP analysis showed that microbiota complexity was high in the first weeks of life, declined to a minimum at 1-2 months of age, and thereafter increased again. Principal component analysis revealed that early samples (1 week-6 months) chiefly differed between individual infants, while 12-month samples were similar between children, but different from the early samples. Our results indicate that T-RFLP has high sensitivity and adequate taxonomic discrimination capacity for analysis of gut microbiota composition, but that both culture and molecular based analysis have limitations and both approaches may be needed to obtain a full picture of the complex gut microbiota.
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| 18. |
- Svensson, Erik, 1959, et al.
(författare)
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Quantitative analyses of mycobacteria in water: adapting methods in clinical laboratories.
- 2011
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Ingår i: Journal of microbiological methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 87:1, s. 114-5
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Tidskriftsartikel (refereegranskat)abstract
- An outbreak of occupational hot tub lung necessitated quantitative analysis of mycobacteria in water samples. We combined procedures for cultivation of mycobacteria in urine and quantitative analyses of dialysis water. Whirlpool spa water samples were analyzed showing promising results. In conclusion, quantitative mycobacterial culture of water is possible by adapting methods routinely used in clinical laboratories.
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| 19. |
- Thierry, S., et al.
(författare)
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A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis
- 2013
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 95:3, s. 357-365
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Tidskriftsartikel (refereegranskat)abstract
- Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2. ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis. © 2013 Elsevier B.V.
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| 20. |
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| 21. |
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| 22. |
- Jaaskelainen, A. J., et al.
(författare)
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Performance of a multiplexed serological microarray for the detection of antibodies against central nervous system pathogens
- 2014
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012. ; 100, s. 27-31
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Tidskriftsartikel (refereegranskat)abstract
- Central nervous system (CNS) infections have multiple potential causative agents for which simultaneous pathogen screening can provide a useful tool. This study evaluated a multiplexed microarray for the simultaneous detection of antibodies against CNS pathogens. The performance of selected microarray antigens for the detection of IgG antibodies against herpes simplex virus 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), adenovirus, Mycoplasma pneumoniae and Borrelia burgdorferi sensu lato, was evaluated using serum sample panels tested with reference assays used in a routine diagnostic laboratory. The microarray sensitivity for HSV-1, HSV-2, VZV, adenovirus and M. pneumonia ranged from 77% to 100%, and the specificity ranged from 74% to 97%. Very variable sensitivities and specificities were found for borrelial antigens of three different VIsE protein IR(6) peptide variants (IR6p1, IR6p2, IR6p4) and three recombinant decorin binding proteins A (DbpA; DbpAla, DbpA91, DbpAG40). For single antigens, good specificity was shown for antigens of IR6p4 and DbpAla (96%), while DbpA91, IR6p1 and IR6p2 were moderately specific (88-92%). The analytical sensitivity of the microarray was dependent on the borrelial IgG concentration of the specimen. The overall performance and technical features of the platform showed that the platform supports both recombinant proteins, whole viruses and peptides as antigens. This study showed diagnostic potential for all six CNS pathogens, including Borrelia burgdorferi sensu lato, using glutaraldehyde based microarray, and further highlighted the importance of careful antigen selection and the requirement for the use of multiple borrelial antigens in order to increase specificity without a major lack of sensitivity. (c) 2014 Elsevier B.V. All rights reserved.
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| 23. |
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