A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.

Ecology of sandflies (Diptera: Psychodidae) in a restricted focus of cutaneous leishmaniasis en Northern Venezuela: I. Description of the study area, catching methods and species composition

A study on the ecology of phlebotomine sandfly fauna in a restricted focus of cutaneous leishmaniasis in northern Venezuela was undertaken in order to investigate the species responsible for the transmission. The study area and catching methods for phlebotomine sandflies are described. A total of 9,061 females and 1,662 males were collected during a year-term study. 12 species of Lutzomya and 1 species of Brumptomya sp. were identified. Absolute and relative abundance and ocurrence for each species were determined. The rel ative occurrence allowed to distinguish the common species, viz. L. panamensis, L. ovallesi, L. gomezi, L. tinidadensis, L. atroclavata, L. cayennensis, L. shannoni and L. olmeca bicolor from the rare species vis., L. punctigeniculata, L. rangeliana, L. evansi and L. dubitans. General comments on the species composition of the sandfly fauna in this locality are made.

Integrative Review: Concepts And Methods Used In Nursing


Integrative review (IR) has an international reputation in nursing research and evidence-based practice. This IR aimed at identifying and analyzing the concepts and methods recommended to undertaking IR in nursing. Nine information resources,including electronic databases and grey literature were searched. Seventeen studies were included. The results indicate that: primary studies were mostly from USA; it is possible to have several research questions or hypotheses and include primary studies in the review from different theoretical and methodological approaches; it is a type of review that can go beyond the analysis and synthesis of findings from primary studies allowing exploiting other research dimensions, and that presents potentialities for the development of new theories and new problems for research. Conclusion: IR is understood as a very complex type of review and it is expected to be developed using standardized and systematic methods to ensure the required rigor of scientific research and therefore the legitimacy of the established evidence.


Spatial and linear correlations between soil and corn

The Technologies setting at Agricultural production system have the main characteristics the vertical productivity, reduced costs, soil physical, chemical and biological improvement to promote production sustainable growth. Thus, the study aimed to determine the variability and the linear and special correlations between the plant and soil attributes in order to select and indicate good representation of soil physical quality for forage productivity. In the growing season of 2006, on the Fazenda Bonança in Pereira Barreto (SP), the productivity of autumn corn forage (FDM) in an irrigated no-tillage system and the soil physical properties were analyzed. The purpose was to study the variability and the linear and spatial correlations between the plant and soil properties, to select an indicator of soil physical quality related to corn forage yield. A geostatistical grid was installed to collect soil and plant data, with 125 sampling points in an area of 2,500 m². The results show that the studied properties did not vary randomly and that data variability was low to very high, with well-defined spatial patterns, ranging from 7.8 to 38.0 m. On the other hand, the linear correlation between the plant and the soil properties was low and highly significant. The pairs forage dry matter versus microporosity and stem diameter versus bulk density were best correlated in the 0-0.10 m layer, while the other pairs - forage dry matter versus macro - and total porosity - were inversely correlated in the same layer. However, from the spatial point of view, there was a high inverse correlation between forage dry matter with microporosity, so that microporosity in the 0-0.10 m layer can be considered a good indicator of soil physical quality, with a view to corn forage yield.

Estimation of water percolation by different methods using TDR

Detailed knowledge on water percolation into the soil in irrigated areas is fundamental for solving problems of drainage, pollution and the recharge of underground aquifers. The aim of this study was to evaluate the percolation estimated by time-domain-reflectometry (TDR) in a drainage lysimeter. We used Darcy's law with K(θ) functions determined by field and laboratory methods and by the change in water storage in the soil profile at 16 points of moisture measurement at different time intervals. A sandy clay soil was saturated and covered with plastic sheet to prevent evaporation and an internal drainage trial in a drainage lysimeter was installed. The relationship between the observed and estimated percolation values was evaluated by linear regression analysis. The results suggest that percolation in the field or laboratory can be estimated based on continuous monitoring with TDR, and at short time intervals, of the variations in soil water storage. The precision and accuracy of this approach are similar to those of the lysimeter and it has advantages over the other evaluated methods, of which the most relevant are the possibility of estimating percolation in short time intervals and exemption from the predetermination of soil hydraulic properties such as water retention and hydraulic conductivity. The estimates obtained by the Darcy-Buckingham equation for percolation levels using function K(θ) predicted by the method of Hillel et al. (1972) provided compatible water percolation estimates with those obtained in the lysimeter at time intervals greater than 1 h. The methods of Libardi et al. (1980), Sisson et al. (1980) and van Genuchten (1980) underestimated water percolation.

Analytical methods for vancomycin determination in biological fluids and in pharmaceuticals

Vancomycin is a glycopeptide antibiotic employed in the treatment of infections caused by certain methicillin-resistant staphylococci. It is indicated also for patients allergic to penicillin or when there is no response to penicillins or cephalosporins. The adequate vancomycin concentration levels in blood serum lies between 5 and 10 mg/L. Higher values are toxic, causing mainly nephrotoxicity and ototoxicity. Various analytical methods are described in the literature: spectrophotometric, immunologic, biologic and chromatographic methods. This paper reviews the main analytical methods for vancomycin determination in biological fluids and in pharmaceutical preparations.

Performance characteristics of high performance liquid chromatography, first order derivative UV spectrophotometry and bioassay for fluconazole determination in capsules

The bioassay, first order derivative UV spectrophotometry and chromatographic methods for assaying fluconazole capsules were compared. They have shown great advantages over the earlier published methods. Using the first order derivative, the UV spectrophotometry method does not suffer interference of excipients. Validation parameters such as linearity, precision, accuracy, limit of detection and limit of quantitation were determined. All methods were linear and reliable within acceptable limits for antibiotic pharmaceutical preparations being accurate, precise and reproducible. The application of each method as a routine analysis should be investigated considering cost, simplicity, equipment, solvents, speed, and application to large or small workloads.

Titrimetric and spectrophotometric assay of bupropion hydrochloride in pharmaceuticals using mercury(II) nitrate

Two simple, rapid and accurate methods for the determination of bupropion hydrochloride (BUP) in pure and in pharmaceutical preparations are described. Both methods are based on the measurement of the chloride of its hydrochloride. In the titrimetric method, the chloride content of bupropion hydrochloride is determined by titrating with mercury(II)nitrate using diphenylcarbazone-bromophenol blue as indicator. Titrimetric method is applicable over a range 2-20 mg of BUP and the reaction stoichiometry is found to be 2:1 (BUP: Hg(NO3)2). The spectrophotometric method involves the addition of a measured excess of mercury(II) nitrate reagent in formate buffer to the drug, and after ensuring the reaction had gone to completion, the unreacted mercury(II) is treated with a fixed amount of diphenylcarbazone, and absorbance measured at 515 nm. The absorbance is found to decrease linearly with increasing concentration of BUP and the calibration curve is linear over 1.0-15.0 µg mL-1 BUP. The proposed methods were successfully applied to the determination of BUP in commercially available dosage forms with good accuracy and precision, and without detectable interference by excipients. The accuracy was further ascertained by placebo blank and synthetic mixture analyses and also by recovery experiments via standard-addition procedure.

Effects of different levels of positive airway pressure on breathing pattern and heart rate variability after coronary artery bypass grafting surgery

The application of continuous positive airway pressure (CPAP) produces important hemodynamic alterations, which can influence breathing pattern (BP) and heart rate variability (HRV). The aim of this study was to evaluate the effects of different levels of CPAP on postoperative BP and HRV after coronary artery bypass grafting (CABG) surgery and the impact of CABG surgery on these variables. Eighteen patients undergoing CABG were evaluated postoperatively during spontaneous breathing (SB) and application of four levels of CPAP applied in random order: sham (3 cmH2O), 5 cmH2O, 8 cmH2O, and 12 cmH2O. HRV was analyzed in time and frequency domains and by nonlinear methods and BP was analyzed in different variables (breathing frequency, inspiratory tidal volume, inspiratory and expiratory time, total breath time, fractional inspiratory time, percent rib cage inspiratory contribution to tidal volume, phase relation during inspiration, phase relation during expiration). There was significant postoperative impairment in HRV and BP after CABG surgery compared to the preoperative period and improvement of DFAα1, DFAα2 and SD2 indexes, and ventilatory variables during postoperative CPAP application, with a greater effect when 8 and 12 cmH2O were applied. A positive correlation (P < 0.05 and r = 0.64; Spearman) was found between DFAα1 and inspiratory time to the delta of 12 cmH2O and SB of HRV and respiratory values. Acute application of CPAP was able to alter cardiac autonomic nervous system control and BP of patients undergoing CABG surgery and 8 and 12 cmH2O of CPAP provided the best performance of pulmonary and cardiac autonomic functions.

Spectral and symbolic analysis of the effect of gender and postural change on cardiac autonomic modulation in healthy elderly subjects

The objective of this study was to use linear and non-linear methods to investigate cardiac autonomic modulation in healthy elderly men and women in response to a postural change from the supine to the standing position. Fourteen men (66.1 ± 3.5 years) and 10 women (65.3 ± 3.3 years) were evaluated. Beat-to-beat heart rate was recorded in the supine and standing positions. Heart rate variability was studied by spectral analysis, including both low (LFnu-cardiac sympathetic modulation (CSM) indicator) and high (HFnu-cardiac vagal modulation (CVM) indicator) frequencies in normalized units as well as the low frequency/high frequency (LF/HF) ratio. Symbolic analysis was performed using the following indexes: 0V% (CSM indicator), 1V% (CSM and CVM indicators), 2LV% (predominantly CVM indicator) and 2ULV% (CVM indicator). Shannon entropy was also calculated. Men presented higher LFnu and LF/HF ratio and lower HFnu and 1V% symbolic index (57.56, 4.14, 40.53, 45.96, respectively) than women (24.60, 0.45, 72.47, 52.69, respectively) in the supine position. Shannon entropy was higher among men (3.53) than among women (3.33) in the standing position, and also increased according to postural change in men (3.25; 3.53). During postural change, the LFnu (24.60; 49.85) and LF/HF ratio (0.45; 1.72) increased, with a concomitant decrease in HFnu (72.47; 47.56) and 2LV% (14.10; 6.95) in women. Women presented increased CSM in response to postural change and had higher CVM and lower CSM than men in the supine position. In conclusion, women in the age range studied presented a more appropriate response to a postural change than men, suggesting that cardiac autonomic modulation may be better preserved in women than in men.

Comparison of methods by TLC and HPTLC for determination of aflatoxin M1 in milk and B1 in eggs

Milk and egg matrixes were assayed for aflatoxin M1 (AFM1) and B1 (AFB1) respectively, by AOAC official and modified methods with detection and quantification by thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC). The modified methods: Blanc followed by Romer, showed to be most appropriate for AFM1 analysis in milk. Both methods reduced emulsion formation, produced cleaner extracts, no streaking spots, precision and accuracy improved, especially when quantification was performed by HPTLC. The use of ternary mixture in the Blanc Method was advantageous as the solvent could extract AFM1 directly from the first stage (extraction), leaving other compounds in the binary mixture layer, avoiding emulsion formation, thus reducing toxin loss. The relative standard deviation (RSD%) values were low, 16 and 7% when TLC and HPTLC were used, with a mean recovery of 94 and 97%, respectively. As far as egg matrix and final extract are concerned, both methods evaluated for AFB1 need further studies. Although that matrix leads to emulsion with consequent loss of toxin, the Romer modified presented a reasonable clean extract (mean recovery of 92 and 96% for TLC and HPTLC, respectively). Most of the methods studied did not performed as expected mainly due to the matrixes high content of triglicerides (rich on saturated fatty acids), cholesterol, carotene and proteins. Although nowadays most methodology for AFM1 is based on HPLC, TLC determination (Blanc and Romer modified) for AFM1 and AFB1 is particularly recommended to those, inexperienced in food and feed mycotoxins analysis and especially who cannot afford to purchase sophisticated (HPLC,HPTLC) instrumentation.

Use of real-time PCR to evaluate two DNA extraction methods from food

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

The influence of extraction methods on composition and antioxidant properties of rice bran oil

AbstractThe current study was employed to assess the influence of the different extraction methods on total tocols, γ-oryzanol content, and antioxidant properties of Chiang Mai Black rice, Mali Red rice, and Suphanburi-1 Brown rice bran oil. Rice bran oil (RBO) was extracted by Hexane, Hot pressed, Cold pressed, and Supercritical Fluid Extraction (SFe) methods. High yield of RBO was extracted by hexane and SFe methods. Total and subgroups of tocols, and γ-oryzanol content were determined by HPLC. The hexane extracted sample accounts for high content of γ-oryzanol and tocols. Besides, all of RBO extracts contain a significantly high amount of γ-tocotrienol. In vitro antioxidant assay results indicated that superior quality of oil was recovered by hexane extraction. The temperature in the extraction process also affects the value of the oil. Superior quality of oil was recovered by hexane extraction, in terms of phytochemical contents and antioxidant properties compared to other tested extraction methods. Further, thorough study of factors compromising the quality and quantity of RBO recovery is required for the development of enhanced functional foods and other related products.