Identification of care needs of patients with and without the use of a classification instrument

Objective: To analyze the agreement and disagreement between the assessments by applying or not a patient classification instrument, and to investigate the association between the agreement and personal and professional characteristics of the evaluators. Method: This is a descriptive exploratory study. 105 patients were hospitalized in a teaching hospital in the state of Sao Paulo, using the kappa statistic (weighted) and the Bootstrap method. Results: The agreement between the assessments were​​: kw 0.87 (instrument x internal evaluator), kw 0.78 (instrument x external evaluator) and kw 0.76 (between evaluators) and the influence of some personal and professional characteristics. The assessments conducted through the use of an instrument contemplated a greater number of areas of care in relation to when the instrument was not applied. Conclusion: The use of this instrument is recommended in order to more effectively identify care needs of patients.




Total fatty acid composition in the characterization and identification of orchid mycorrhizal fungi Epulorhiza spp.

Rhizoctonia-like fungi are the main mycorrhizal fungi in orchid roots. Morphological characterization and analysis of conserved sequences of genomic DNA are frequently employed in the identification and study of fungi diversity. However, phytopathogenic Rhizoctonia-like fungi have been reliably and accurately characterized and identified through the examination of the fatty acid composition. To evaluate the efficacy of fatty acid composition in characterizing and identifying Rhizoctonia-like mycorrhizal fungi in orchids, three Epulorhiza spp. mycorrhizal fungi from Epidendrum secundum, two unidentified fungi isolated from Epidendrum denticulatum, and a phytopathogenic fungus, Ceratorhiza sp. AGC, were grouped based on the profile of their fatty acids, which was assessed by the Euclidian and Mahalanobis distances and the UPGMA method. Dendrograms distinguished the phytopathogenical isolate of Ceratorhiza sp. AGC from the mycorrhizal fungi studied. The symbionts of E. secundum were grouped into two clades, one containing Epulorhiza sp.1 isolates and the other the Epulorhiza sp.2 isolate. The similarity between the symbionts of E. denticulatum and Epulorhiza spp. fungi suggests that symbionts found in E. denticulatum may be identified as Epulorhiza. These results were corroborated by the analysis of the rDNA ITS region. The dendrogram constructed based on the Mahalanobis distance differentiated the clades most clearly. Fatty acid composition analysis proved to be a useful tool for characterizing and identifying Rhizoctonia-like mycorrhizal fungi.

Molecular Identification of Trichoderma spp. in Garlic and Onion Fields and In Vitro Antagonism Trials on Sclerotium cepivorum

ABSTRACT Trichoderma species are non-pathogenic microorganisms that protect against fungal diseases and contribute to increased crop yields. However, not all Trichoderma species have the same effects on crop or a pathogen, whereby the characterization and identification of strains at the species level is the first step in the use of a microorganism. The aim of this study was the identification – at species level – of five strains of Trichoderma isolated from soil samples obtained from garlic and onion fields located in Costa Rica, through the analysis of the ITS1, 5.8S, and ITS2 ribosomal RNA regions; as well as the determination of their individual antagonistic ability over S. cepivorum Berkeley. In order to distinguish the strains, the amplified products were analyzed using MEGA v6.0 software, calculating the genetic distances through the Tamura-Nei model and building the phylogenetic tree using the Maximum Likelihood method. We established that the evaluated strains belonged to the species T. harzianum and T. asperellum; however it was not possible to identify one of the analyzed strains based on the species criterion. To evaluate their antagonistic ability, the dual culture technique, Bell’s scale, and the percentage inhibition of radial growth (PIRG) were used, evidencing that one of the T. asperellum isolates presented the best yields under standard, solid fermentation conditions.

Optimal use of SSR markers for varietal identification of upland cotton

Abstract: The objective of this work was to identify polymorphic simple sequence repeat (SSR) markers for varietal identification of cotton and evaluation of the genetic distance among the varieties. Initially, 92 SSR markers were genotyped in 20 Brazilian cotton cultivars. Of this total, 38 loci were polymorphic, two of which were amplified by one primer pair; the mean number of alleles per locus was 2.2. The values of polymorphic information content (PIC) and discrimination power (DP) were, on average, 0.374 and 0.433, respectively. The mean genetic distance was 0.397 (minimum of 0.092 and maximum of 0.641). A panel of 96 varieties originating from different regions of the world was assessed by 21 polymorphic loci derived from 17 selected primer pairs. Among these varieties, the mean genetic distance was 0.387 (minimum of 0 and maximum of 0.786). The dendrograms generated by the unweighted pair group method with arithmetic average (UPGMA) did not reflect the regions of Brazil (20 genotypes) or around the world (96 genotypes), where the varieties or lines were selected. Bootstrap resampling shows that genotype identification is viable with 19 loci. The polymorphic markers evaluated are useful to perform varietal identification in a large panel of cotton varieties and may be applied in studies of the species diversity.

Identification of plantlet genetic origin in polyembryonic mango (Mangifera indica, L.) cv. Rosinha seeds using RAPD markers

Mango can be propagated by seeds or by grafting. For commercial purpose, grafting is the most appropriate method because it maintains the genetic characters from the propagated variety. To obtain grafted mango it is important to use polyembryonic varieties as rootstock since they produce a zygotic and many nucellar plantlets. The nucellar plantlets maintain the genetics of the mother-plant thus, are preferred for grafting since they supposedly give more uniformity to the orchard. In general, nurserymen use the most vigorous plantelet to graft, believing that they are nucellar. But, orchard disuniformities on height and yield are very common among mango trees of commercial orchards in Northeast region. The objective of this paper was to identify the genetical origin of plantlets from polyembryonic seeds of Rosinha variety using Random Amplified Polymorphic DNA (RAPD) markers. Moreover, the position of the zygotic embryo and the percentage of the vigorous zygotic and nucellar plantlets was also determined. It was obtained an elevated taxa of vigorous zygotic plantlets which possibly explains the disuniformity on height of trees at commercial mango orchards.

HPLC-DAD based method for the quantification of flavonoids in the hydroethanolic extract of Tonina fluviatilis Aubl. (Eriocaulaceae) and their radical scavenging activity

This article describes the isolation and identification of flavonoids in the hydroethanolic extract of the aerial parts from Tonina fluviatilis and evaluation of their antiradical activity. A method based on HPLC-DAD was developed and validated for detecting and quantifying flavonoids in hydroethanolic extracts. The flavonoids identified and quantified in the extract were 6,7-dimethoxyquercetin-3-O-β-D-glucopyranoside (1), 6-hydroxy-7-methoxyquercetin-3-O-β-D-glucopyranoside (2), and 6-methoxyquercetin-3-O-β-D-glucopyranoside (3). The developed method presented good validation parameters, showing that the results obtained are consistent and can be used in ensuring the quantification of these constituents in the extracts. Compounds 2 and 3 showed strong antiradical activity when compared with the positive controls (quercetin and gallic acid).

Spot-test identification and rapid quantitative sequential analysis of dipyrone

A qualitative spot-test and tandem quantitative analysis of dipyrone in the bulk drug and in pharmaceutical preparations is proposed. The formation of a reddish-violet color indicates a positive result. In sequence a quantitative procedure can be performed in the same flask. The quantitative results obtained were statistically compared with those obtained with the method indicated by the Brazilian Pharmacopoeia, using the Student's t and the F tests. Considering the concentration in a 100 µL aliquot, the qualitative visual limit of detection is about 5×10-6 g; instrumental LOD ≅ 1.4×10-4 mol L-1 ; LOQ ≅ 4.5×10-4 mol L-1.

Identification of reciprocal hybrids in citrus by the broadness of the leaf petiole wing

Broadness of leaf petiole wing (WB) was investigated as a morphological marker for screening hybrids of the very narrow-winged species Citrus limonia and C. sunki with broad-winged species C. aurantium and C. sinensis. Controlled polinizations produced over 500 reciprocal hybrids with potential in the ongoing rootstock breeding program identified by the Pgi-1 and Prxa-1 isozyme loci. Measurement ratios WB/leaf length, WB/leaf broadness and WB/petiole length identified 86 to 91% of the reciprocal hybrids produced. However, visual classification of WB was an equally efficient but much easier and faster method. It can be very useful in breeding programs when large number of plants have to be screened or when isozyme, RFLP or RAPD laboratories are not available.

Identification of clonally rearranged T-cell receptor beta chain genes in HTLV-I carriers as a potential instrument for early detection of neoplasia

We analyzed the genetic recombination pattern of the T-cell receptor beta-chain gene (TCR-beta) in order to identify clonal expansion of T-lymphocytes in 17 human T-lymphotropic virus type I (HTLV-I)-positive healthy carriers, 7 of them with abnormal features in the peripheral blood lymphocytes. Monoclonal or oligoclonal expansion of T-cells was detected in 5 of 7 HTLV-I-positive patients with abnormal lymphocytes and unconfirmed diagnosis by using PCR amplification of segments of TCR-beta gene, in a set of reactions that target 102 different variable (V) segments, covering all members of the 24 V families available in the gene bank, including the more recently identified segments of the Vbeta-5 and Vbeta-8 family and the two diversity beta segments. Southern blots, the gold standard method to detect T-lymphocyte clonality, were negative for all of these 7 patients, what highlights the low sensitivity of this method that requires a large amount of very high quality DNA. To evaluate the performance of PCR in the detection of clonality we also analyzed 18 leukemia patients, all of whom tested positive. Clonal expansion was not detected in any of the negative controls or healthy carriers without abnormal lymphocytes. In conclusion, PCR amplification of segments of rearranged TCR-beta is reliable and highly suitable for the detection of small populations of clonal T-cells in asymptomatic HTLV-I carriers who present abnormal peripheral blood lymphocytes providing an additional instrument for following up these patients with potentially higher risk of leukemia.

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line

Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45% in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5%, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27%, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.

Identification of volatile compounds in thinning discards from plum trees (Prunus salicina Lindl.) cultivar Harry Pickstone

Plum (Prunus salicina Lindl. cv. Harry Pickstone), a China indigenous fruit, is widely produced and consumed in countries such as Japan and Brazil. The practice of thinning is common in horticulture and the fruits removed are discarded as waste. Like the great majority of vegetables, these thinning discards also contain essential oils which have not been investigated until the present time. The extraction of the plum thinning discards volatile oil, through the hydrodistillation method, produced a yield of 0.06% (m/m) and a total of 21 components were identified, with 11 of them being responsible for 72,9% of the total oil composition. The major compounds determined through GC and GC-MS were Z-α-bisabolene (13.7%), n-hexadecanoic acid (12.7%), phytol (12.7%), and β-caryophyllene (10.4%).

Development of a rapid method for the determination of antibiotic residues in honey using UPLC-ESI-MS/MS

Abstract An accurate, reliable and fast multianalyte/multiclass ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the simultaneous analysis of 23 pharmaceuticals, belonging to different classes amphenicols, sulfonamides, tetracyclines, in honey samples. The method developed consists of ultrasonic extraction followed by UPLC–ESI–MS/MS with electrospray ionization in both positive mode and negative mode. The influence of the extraction solvents and mobile phase composition on the sensitivity of the method, and the optimum conditions for sample weight and extraction temperature in terms of analyte recovery were extensively studied. The identification of antibiotics is fulfilled by simultaneous use of chromatographic separation using an Acquity BEH C18 (100 mm x 2.1 mm, 1.7 µm) analytical column with a gradient elution of mobile phases and tandem mass spectrometry with an electrospray ionization. Finally, the method developed was applied to the determination of target analytes in honey samples obtained from the local markets and several beekeepers in Muğla, Turkey. Ultrasonic-extraction of pharmaceuticals from honey samples is a well-established technique by UPLC–ESI–MS/MS, the uniqueness of this study lies in the simultaneous determination of a remarkable number of compounds belonging to 23 drug at the sub-nanogram per kilogram level.