An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests. Organophosphate compounds are used in agro-systems, and in programs to control pathogen vectors. Because they are continuously applied, organophosphates often reach water sources and may have an impact on aquatic life. The effects of acute and chronic exposure to the organophosphate insecticide malathion on the midge Chironomus sancticaroli are evaluated. To that end, three biochemical biomarkers, acetylcholinesterase (AChE), alpha (EST-α) and beta (EST-β) esterase were used. Acute bioassays with five concentrations of malathion, and chronic bioassays with two concentrations of malathion were carried out. In the acute exposure test, AChE, EST-α and EST-β activities declined by 66, 40 and 37%, respectively, at 0.251 µg L-1 and more than 80% at 1.37, 1.96 and 2.51 µg L-1. In chronic exposure tests, AChE and EST-α activities declined by 28 and 15% at 0.251 µg L-1. Results of the present study show that low concentrations of malathion can influence larval metabolism, indicating high toxicity for Chironomus sancticaroli and environmental risk associated with the use of organophosphates.

Behavioral avoidance tests to evaluate effects of cattle slurry and dairy sludge application to soil¹

The application of organic wastes to agricultural soils is not risk-free and can affect soil invertebrates. Ecotoxicological tests based on the behavioral avoidance of earthworms and springtails were performed to evaluate effects of different fertilization strategies on soil quality and habitat function for soil organisms. These tests were performed in soils treated with: i) slurry and chemical fertilizers, according to the conventional fertilization management of the region, ii) conventional fertilization + sludge and iii) unfertilized reference soil. Both fertilization strategies contributed to soil acidity mitigation and caused no increase in soil heavy metal content. Avoidance test results showed no negative effects of these strategies on soil organisms, compared with the reference soil. However, results of the two fertilization managements differed: Springtails did not avoid soils fertilized with dairy sludge in any of the tested combinations. Earthworms avoided soils treated with sludge as of May 2004 (DS1), when compared with conventional fertilization. Possibly, the behavioral avoidance of earthworms is more sensitive to soil properties (other than texture, organic matter and heavy metal content) than springtails

Hydrological processes obtained on the plot scale under four simulated rainfall tests during the cycle of different crop systems

The cropping system influences the interception of water by plants, water storage in depressions on the soil surface, water infiltration into the soil and runoff. The aim of this study was to quantify some hydrological processes under no tillage cropping systems at the edge of a slope, in 2009 and 2010, in a Humic Dystrudept soil, with the following treatments: corn, soybeans, and common beans alone; and intercropped corn and common bean. Treatments consisted of four simulated rainfall tests at different times, with a planned intensity of 64 mm h-1 and 90 min duration. The first test was applied 18 days after sowing, and the others at 39, 75 and 120 days after the first test. Different times of the simulated rainfall and stages of the crop cycle affected soil water content prior to the rain, and the time runoff began and its peak flow and, thus, the surface hydrological processes. The depth of the runoff and the depth of the water intercepted by the crop + soil infiltration + soil surface storage were affected by the crop systems and the rainfall applied at different times. The corn crop was the most effective treatment for controlling runoff, with a water loss ratio of 0.38, equivalent to 75 % of the water loss ratio exhibited by common bean (0.51), the least effective treatment in relation to the others. Total water loss by runoff decreased linearly with an increase in the time that runoff began, regardless of the treatment; however, soil water content on the gravimetric basis increased linearly from the beginning to the end of the rainfall.

Static tests of constructive tires using hydraulic press on the soil

ABSTRACT The power consumption and load capacity of agricultural machines have grown and the effects of pressure on the soil by tires have been still little investigated. In concern with sustainable development, the relationship machine-tire-soil must be in balance to give more consistency on the best use of tires for a given load. This study aimed to evaluate four tires of two constructive types, the bias belted tires and radial tires, both with respective rim diameters of 22.5 and 26.5 inches with variables measuring the footprint, elastic deformation, sinkage and resistance to penetration. A hydraulic press with an attachment shaft for tire mounting and a box of soil in which the tire has been imposed on a load of 53.00 kN using nominal pressures recommended by the tire manufacturer. The radial construction tire with rim diameter of 26.5 inches obtained less sinkage and resistance to penetration; however, greater elastic deformation and footprint compared to other tires. The bias-belted tire with 22.5-inch rim presented the highest resistance to penetration and the lowest elastic deformation.

Quality Indicators of Cervical Cytopathology Tests in the Public Service in Minas Gerais, Brazil

Objective The objective of this study is to assess the performance of cytopathology laboratories providing services to the Brazilian Unified Health System (Sistema Único de Saúde - SUS) in the State of Minas Gerais, Brazil. Methods This descriptive study uses data obtained from the Cervical Cancer Information System from January to December 2012. Three quality indicators were analyzed to assess the quality of cervical cytopathology tests: positivity index, percentage of atypical squamous cells (ASCs) in abnormal tests, and percentage of tests compatiblewith high-grade squamous intraepithelial lesions (HSILs). Laboratories were classified according to their production scale in tests per year≤5,000; from 5,001 to 10,000; from 10,001 to 15,000; and 15,001. Based on the collection of variables and the classification of laboratories according to production scale, we created and analyzed a database using Microsoft Office Excel 97-2003. Results In the Brazilian state of Minas Gerais, 146 laboratories provided services to the SUS in 2012 by performing a total of 1,277,018 cervical cytopathology tests. Half of these laboratories had production scales≤5,000 tests/year and accounted for 13.1% of all tests performed in the entire state; in turn, 13.7% of these laboratories presented production scales of > 15,001 tests/year and accounted for 49.2% of the total of tests performed in the entire state. The positivity indexes of most laboratories providing services to the SUS in 2012, regardless of production scale, were below or well below recommended limits. Of the 20 laboratories that performed more than 15,001 tests per year, only three presented percentages of tests compatible with HSILs above the lower limit recommended by the Brazilian Ministry of Health. Conclusion The majority of laboratories providing services to the SUS in Minas Gerais presented quality indicators outside the range recommended by the Brazilian Ministry of Health.

Predictive Capability of HPV and Pap Tests in Screening for Cervical Cancer over a Three-Year Follow-up

Purpose To compare the predictive capability of HPV and Pap smear tests for screening pre-cancerous lesions of the cervix over a three-year follow-up, in a population of users of the Brazilian National Health System (SUS). Methods This is a retrospective cohort study of 2,032 women with satisfactory results for Pap smear and HPV tests using second-generation hybrid capture,made in a previous study. We followed them for 36 months with data obtained from medical records, the Cervix Cancer Information System (SISCOLO), and the Mortality Information System (SIM). The outcome was a histological diagnosis of cervical intraepithelial neoplasia grade 2 or more advanced lesions (CIN2ş). We constructed progression curves of the baseline test results for the period, using the Kaplan-Meier method, and estimated sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ratios for each test. Results A total of 1,440 women had at least one test during follow-up. Progression curves of the baseline test results indicated differences in capability to detect CIN2ş (p < 0.001) with significantly greater capability when both tests were abnormal, followed by only a positive HPV test. The HPV test was more sensitive than the Pap smear (88.7% and 73.6%, respectively; p < 0.05) and had a better negative likelihood ratio (0.13 and 0.30, respectively). Specificity and positive likelihood ratio of the tests were similar. Conclusions These findings corroborate the importance of HPV test as a primary cervical cancer screening.

Reference values for lung function tests: I. Static volumes

Static lung volume (LV) measurements have a number of clinical and research applications; however, no previous studies have provided reference values for such tests using a healthy sample of the adult Brazilian population. With this as our main purpose, we prospectively evaluated 100 non-smoking subjects (50 males and 50 females), 20 to 80 years old, randomly selected from more than 8,000 individuals. Gender-specific linear prediction equations were developed by multiple regression analysis with total lung capacity (TLC), functional residual capacity (FRC), residual volume (RV), RV/TLC ratio and inspiratory capacity (IC) as dependent variables, and with age, height, weight, lean body mass and indexes of physical fitness as independent ones. Simpler demographic and anthropometric variables were as useful as more complex measurements in predicting LV values, independent of gender and age (R2 values ranging from 0.49 to 0.78, P<0.001). Interestingly, prediction equations from North American and European studies overestimated the LV at low volumes and underestimated them at high volumes (P<0.05). Our results, therefore, provide a more appropriate frame of reference to evaluate the normalcy of static lung volume values in Brazilian males and females aged 20 to 80 years.

Reference values for lung function tests: II. Maximal respiratory pressures and voluntary ventilation

The strength of the respiratory muscles can be evaluated from static measurements (maximal inspiratory and expiratory pressures, MIP and MEP) or inferred from dynamic maneuvers (maximal voluntary ventilation, MVV). Although these data could be suitable for a number of clinical and research applications, no previous studies have provided reference values for such tests using a healthy, randomly selected sample of the adult Brazilian population. With this main purpose, we prospectively evaluated 100 non-smoking subjects (50 males and 50 females), 20 to 80 years old, selected from more than 8,000 individuals. Gender-specific linear prediction equations for MIP, MEP and MVV were developed by multiple regression analysis: age and, secondarily, anthropometric measurements explained up to 56% of the variability of the dependent variables. The most cited previous studies using either Caucasian or non-Caucasian samples systematically underestimated the observed values of MIP (P<0.05). Interestingly, the self-reported level of regular physical activity and maximum aerobic power correlates strongly with both respiratory and peripheral muscular strength (knee extensor peak torque) (P<0.01). Our results, therefore, provide a new frame of reference to evaluate the normalcy of some useful indexes of respiratory muscle strength in Brazilian males and females aged 20 to 80.

Reference values for lung function tests: III. Carbon monoxide diffusing capacity (transfer factor)

Carbon monoxide diffusing capacity (DLCO) or transfer factor (TLCO) is a particularly useful test of the appropriateness of gas exchange across the lung alveolocapillary membrane. With the purpose of establishing predictive equations for DLCO using a non-smoking sample of the adult Brazilian population, we prospectively evaluated 100 subjects (50 males and 50 females aged 20 to 80 years), randomly selected from more than 8,000 individuals. Gender-specific linear prediction equations were developed by multiple regression analysis with single breath (SB) absolute and volume-corrected (VA) DLCO values as dependent variables. In the prediction equations, age (years) and height (cm) had opposite effects on DLCOSB (ml min-1 mmHg-1), independent of gender (-0.13 (age) + 0.32 (height) - 13.07 in males and -0.075 (age) + 0.18 (height) + 0.20 in females). On the other hand, height had a positive effect on DLCOSB but a negative one on DLCOSB/VA (P<0.01). We found that the predictive values from the most cited studies using predominantly Caucasian samples were significantly different from the actually measured values (P<0.05). Furthermore, oxygen uptake at maximal exercise (VO2max) correlated highly to DLCOSB (R = 0.71, P<0.001); this variable, however, did not maintain an independent role to explain the VO2max variability in the multiple regression analysis (P>0.05). Our results therefore provide an original frame of reference for either DLCOSB or DLCOSB/VA in Brazilian males and females aged 20 to 80 years, obtained from the standardized single-breath technique.

Tomato and garlic by gavage modulate 7,12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress in mice

Chemoprotection by dietary agents is a promising strategy for cancer prevention. The aim of the present study was to evaluate the combined effect of tomato and garlic against 7,12-dimethylbenz- [a]anthracene (DMBA)-induced genetic damage and oxidative stress in 12-14-week-old male Swiss albino mice. The animals were randomized into experimental and control groups and divided into eight groups of five animals each. Group 1 animals were injected intraperitoneally with 35 mg/kg body weight DMBA suspended in peanut oil as a single dose. Groups 2-4 animals received tomato (500 mg/kg body weight), garlic (125 mg/kg body weight) and a combination of tomato and garlic for 5 days by gavage, respectively, followed by DMBA 1.5 h after the final feeding. The doses of tomato and garlic correspond to the average human daily consumption. Animals in groups 5, 6 and 7 received tomato alone, garlic alone and tomato + garlic combination, respectively, for 5 days. Group 8 animals received the same volume of water and served as control. The incidence of bone marrow micronuclei and the extent of lipid peroxidation and the concentrations of antioxidants glutathione, glutathione peroxidase and glutathione-S-transferase were measured in the liver, 48 h after DMBA exposure. Increased frequency of micronuclei and enhanced lipid peroxidation accompanied by compromised antioxidant defenses were observed in DMBA-treated animals. Although pretreatment with tomato or garlic significantly reduced the frequency of DMBA-induced bone marrow micronuclei, the combination of tomato and garlic exhibited more profound effect in inhibiting DMBA-induced genotoxicity and oxidative stress. We suggest that a broad spectrum of antimutagenic and anticlastogenic effects can be achieved through an effective combination of functional foods such as tomato and garlic.

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

The inactivation of the basolateral nucleus of the rat amygdala has an anxiolytic effect in the elevated T-maze and light/dark transition tests

Pharmacological evidence indicates that the basolateral nucleus of the amygdala (BLA) is involved in the mediation of inhibitory avoidance but not of escape behavior in the elevated T-maze test. These defensive responses have been associated with generalized anxiety disorder (GAD) and panic disorder, respectively. In the present study, we determined whether the BLA plays a differential role in the control of inhibitory avoidance and escape responses in the elevated T-maze. Male Wistar rats (250-280 g, N = 9-10 in each treatment group) were pre-exposed to one of the open arms of the maze for 30 min and 24 h later tested in the model after inactivation of the BLA by a local injection of the GABA A receptor agonist muscimol (8 nmol in 0.2 µL). It has been shown that a prior forced exposure to one of the open arms of the maze, by shortening latencies to withdrawal from the open arm during the test, improves the escape task as a behavioral index of panic. The effects of muscimol in the elevated T-maze were compared to those caused by this GABA agonist in the avoidance reaction generated in the light/dark transition test. This defensive behavior has also been associated with GAD. In the elevated T-maze, intra-BLA injection of muscimol impaired inhibitory avoidance (control: 187.70 ± 14.90 s, muscimol: 37.10 ± 2.63 s), indicating an anxiolytic effect, without interfering with escape performance. The drug also showed an anxiolytic effect in the light/dark transition test as indicated by the increase in the time spent in the lighted compartment (control: 23.50 ± 2.45 s, muscimol: 47.30 ± 4.48 s). The present findings point to involvement of the BLA in the modulation of defensive responses that have been associated with GAD.

Dose-response effects of systemic anandamide administration in mice sequentially submitted to the open field and elevated plus-maze tests

The endocannabinoid system is involved in the control of many physiological functions, including the control of emotional states. In rodents, previous exposure to an open field increases the anxiety-like behavior in the elevated plus-maze. Anxiolytic-like effects of pharmacological compounds that increase endocannabinoid levels have been well documented. However, these effects are more evident in animals with high anxiety levels. Several studies have described characteristic inverted U-shaped dose-response effects of drugs that modulate the endocannabinoid levels. However, there are no studies showing the effects of different doses of exogenous anandamide, an endocannabinoid, in animal models of anxiety. Thus, in the present study, we determined the dose-response effects of exogenous anandamide at doses of 0.01, 0.1, and 1.0 mg/kg in C57BL/6 mice (N = 10/group) sequentially submitted to the open field and elevated plus-maze. Anandamide was diluted in 0.9% saline, ethyl alcohol, Emulphor® (18:1:1) and administered ip (0.1 mL/10 g body weight); control animals received the same volume of anandamide vehicle. Anandamide at the dose of 0.1 mg/kg (but not of 0.01 or 1 mg/kg) increased (P < 0.05) the time spent and the distance covered in the central zone of the open field, as well as the exploration of the open arms of the elevated plus-maze. Thus, exogenous anandamide, like pharmacological compounds that increase endocannabinoid levels, promoted a characteristic inverted U-shaped dose-response effect in animal models of anxiety. Furthermore, anandamide (0.1 mg/kg) induced an anxiolytic-like effect in the elevated plus-maze (P < 0.05) after exposing the animals to the open field test.