Organic and total mercury determination in sediments by cold vapor atomic absorption spectrometry: methodology validation and uncertainty measurements

The purpose of the present study was to validate a method for organic Hg determination in sediment. The procedure for organic Hg was adapted from literature, where the organomercurial compounds were extracted with dichloromethane in acid medium and subsequent destruction of organic compounds by bromine chloride. Total Hg was performed according to 3051A USEPA methodology. Mercury quantification for both methodologies was then performed by CVAAS. Methodology validation was verified by analyzing certified reference materials for total Hg and methylmercury. The uncertainties for both methodologies were calculated. The quantification limit of 3.3 µg kg-1 was found for organic Hg by CVAAS.

QUANTIFICATION OF THE POPULATION AND PHAGOCYTARY ACTIVITY OF HEMOCYTES OF RESISTANT AND SUSCEPTIBLE STRAINS OF Biomphalaria glabrata AND Biomphalaria tenagophila INFECTED WITH Schistosoma mansoni

Among the determinant factors in the resistance and susceptibility of Biomphalaria to Schistosoma mansoni, hemocytes play an important role. Aiming at studying S. mansoni/Biomphalaria interactions related to hemocytes, the first step is certainly connected with the standardization of this cell population in uninfected Biomphalaria. In this way, quantification of this cell population in hemolymph, as well as its phagocitary capacity, have been determined for the first time. Furthermore, using susceptible and resistant strains of B. glabrata and B. tenagophila, the hemocytegram and phagocytary capacity of hemocytes after infection with S. mansoni were determined too. Resistant and susceptible strains of B.glabrata (BA and BH, respectively), as well as resistant and susceptible strains of B. tenagophila (Taim and CF, respectively) were infected with 10 miracidia of the LE and SJ strains of S. mansoni, respectively. These infected snails and respective uninfected controls were assessed in relation to the number of circulating hemocytes and alteration in the phagocytary capacity, by using Zymozan and MTT. Reading was taken by means of a spectrophotometer at 5 hours and 1,2,5,10,20 and 30 days after infection. The results showed a decrease in population of the circulating phagocytary cells, 5 hours after infection. One day post-infection, the circulating cells of the susceptible snails showed an increased metabolic activity, but the same event could not be observed in the resistant strains. In the subsequent observation periods, significant differences among the strains studied could not be observed until the end of the experiment

Quantification of HIV-1 viral RNA in the blood in needles used for venous puncture in HIV-infected individuals

INTRODUCTION: Occupational HIV infection among healthcare workers is an important issue in exposures involving blood and body fluids. There are few data in the literature regarding the potential and the duration of infectivity of HIV type 1 (HIV-1) in contaminated material under adverse conditions. METHODS: We quantified HIV-1 viral RNA in 25×8mm calibre hollow-bore needles, after punctures, in 25 HIV-1-infected patients selected during the sample collection. All of the patients selected were between the ages of 18 and 55. Five samples were collected from 16 patients: one sample for the immediate quantification of HIV-1 RNA in the plasma and blood samples from the interior of 4 needles to be analyzed at 0h, 6h, 24h, and 72h after collection. In nine patients, another test was carried out in the blood from one additional needle, in which HIV-1 RNA was assessed 168h after blood collection. The method used to assess HIV-1 RNA was nucleic acid sequence-based amplification. RESULTS: Up to 7 days after collection, HIV-1 RNA was detected in all of the needles. The viral RNA remained stable up to 168h, and there were no statistically significant differences among the needle samples. CONCLUSIONS: Although the infectivity of the viral material in the needles is unknown, the data indicate the need to re-evaluate the practices in cases of occupational accidents in which the source is not identified.

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.

Quantification of Trypanosoma cruzi in the heart, lymph nodes and liver of experimentally inffected mice, using limiting dilution analysis

Limiting dilution analysis was used to quantify Trypanosoma cruzi in the lymph nodes, liver and heart of Swiss and C57 B1/10 mice. The results showed that, in Swiss and B1/10 mice infected with T. cruzi Y strain, the number of parasites/mg of tissue increased during the course of the infection in both types of mice, although a grater number of parasites were observed in heart tissue from Swiss mice than from B1/10. With regard to liver tissue, it was observed that the parasite load in the initial phase of infection was higher than in heart. In experiments using T. cruzi Colombian strain, the parasite load in the heart of Swiss and B1/10 mice increased relatively slowly, although high levels of parasitization were nonetheless observable by the end of the infection. As for the liver and lymph nodes, the concentration of parasites was lower over the entire course of infection than in heart. Both strains thus maintained their characteristic tissue tropisms. The limiting dilution assay (LDA) proved to be an appropriate method for more precise quantification of T. cruzi, comparing favorably with other direct microscopic methods that only give approximate scores.

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples.

Quantification of the genetic change in the transition of Rhodnius pallescens Barber, 1932 (Hemiptera: Reduviidae) from field to laboratory

Previous studies have reported genetic differences between wild-caught sylvatic, domestic and laboratory pop-ulations of several Triatominae species. The differences between sylvatic and laboratory colonies parallel are similar to the differences observed between sylvatic and domestic populations. Laboratory colonies are frequently used as references for field populations, but the consequences of founder events on the genetic makeup of laboratory or domestic populations are rarely quantified. Our goal was to quantify the genetic change in Rhodnius pallescens populations artificially submitted to founder effects via laboratory colonization. We compared the genetic makeup of two sylvatic populations and their laboratory descendants using a panel of 10 microsatellite markers. Both sylvatic populations were initially collected from palm trees, but the colonies differed in the number of founder insects and amount of time kept in the laboratory. We evaluated allelic polymorphism, differences between expected and observed heterozygosity, estimates of population differentiation (Fst) and inbreeding (Fis, Fit) and cluster analyses based on Nei's distances. We found a unique genetic structure for each sample population, with significant differentiation between the field insects and each of the laboratory generations. These analyses showed strong founder effects and showed that genetic drift had led to a genetic equilibrium over several generations of isolation. Our results suggest that laboratory colonies of R. pallescens have a different genetic structure than their wild relatives and similar processes likely affect other Triatominae laboratory stocks.

Real-time PCR-based quantification of Toxoplasma gondii in tissue samples of serologically positive outdoor chickens

This study aimed to quantify Toxoplasma gondii in tissue samples of serologically positive chickens using real-time polymerase chain reaction (PCR). Of 65 chickens evaluated, 28 were positive for T. gondii antibodies. Brain and heart samples were collected from 26 seropositive chickens and DNA was extracted using Trizol® and amplified using real-time PCR with SYBR® Green. Parasite DNA was detected in 24 of the 26 samples analyzed; the number of positive tissue samples and the parasite quantity did not differ between tissue types. The results confirmed the analytical sensitivity of parasite detection in chicken tissue samples and demonstrated the possibility of using other molecular systems for genotypic analysis.

Hepatitis C virus quantification in serum and saliva of HCV-infected patients

The hepatitis C virus (HCV) can be detected in blood and other bodily fluids, such as saliva, semen and gastric juices. The aim of this study was to compare the HCV viral loads in the serum and saliva of infected patients. Twenty-nine patients with detectable HCV RNA in their serum and saliva were included in this study. The HCV viral loads were determined through quantitative real-time polymerase chain reactions. The median viral RNA levels were 5.78 log10 copies in the serum and 3.32 log10 copies in the saliva. We observed that the salivary HCV viral load was significantly lower than the viral load in the serum. Further studies are required to understand the role of saliva in the diagnosis, management and potential transmission of HCV.

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

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.

Quality uncertainty and allocation of decision rights in the European protected designation of origin

The paper considers some issue in the governance of the European Protected Designation of Origin (PDO). The PDO systems are the outcomes of both farmers and consumers expectations and connect the valorisation of the agricultural and rural resources of given territories to the quality of typical products. A critical point in the governance of the PDO systems is represented by the connection between the quality strategies and the uncertainty. The paper argues that the PDO systems can be thought of as strictly coordinated subsystems in which the ex post governance play a critical role in coping with quality uncertainty. The study suggests that the society's inducements given raise to complex organizational systems in which the allocation of decision rights to PDO collective organizations play a major role. The empirical analysis is carried out by examining ten Italian PDO systems in order to identify the decision rights allocated.

Entrepreneurial decision-making using the knightian uncertainty approach

The article discusses the behavioral aspects that affect the entrepreneurs' decision making under the Knightian uncertainty approach. Since the profit arising from entrepreneurial activity represents the reward of an immeasurable and subjective risk, it has been hypothesized that innovative entrepreneurs have excessive optimism and confidence, which leads them to invest in high-risk activities. A behavioral model of decision making under uncertainty is used to test the hypothesis of overconfidence. This model is based on Bayesian inference, which allows us to model the assumption that these entrepreneurs are overconfident. We conclude that, under the hypothesis of overconfidence, these entrepreneurs decide to invest, despite the fact that the expected utility model indicates the contrary. This theoretical finding could explain why there are a large number of business failures in the first years of activity.

Spectroscopic quantification of soil phosphorus forms by 31p-nmr after nine years of organic or mineral fertilization

Long-standing applications of mineral fertilizers or types of organic wastes such as manure can cause phosphorus (P) accumulation and changes in the accumulated P forms in the soil. The objective of this research was to evaluate the forms of P accumulated in soils treated with mineral fertilizer or different types of manure in a long-term experiment. Soil was sampled from the 0-5 cm layer of plots fertilized with five different nutrient sources for nine years: 1) control without fertilizer; 2) mineral fertilizer at recommended rates for local conditions; 3) 5 t ha-1 year-1 of moist poultry litter; 4) 60 m³ ha-1 year-1 of liquid cattle manure and 5) 40 m³ ha-1 year-1 of liquid swine manure. The 31P-NMR spectra of soil extracts detected the following P compounds: orthophosphate, pyrophosphate, inositol phosphate, glycerophosphate, and DNA. The use of organic or mineral fertilizer over nine years did not change the soil P forms but influenced their concentration. Fertilization with mineral or organic fertilizers stimulated P accumulation in inorganic forms. Highest inositol phosphate levels were observed after fertilization with any kind of manure and highest organic P concentration in glycerophosphate form in after mineral or no fertilization.