Comparison of the efficacy of biodegradable and non-biodegradable scintillation liquids on the counting of tritium- and [14C]-labeled compounds

The widespread use of ³H and 14C in research has generated a large volume of waste mixed with scintillation liquid, requiring an effective control and appropriate storage of liquid radioactive waste. In the present study, we compared the efficacy of three commercially available scintillation liquids, Optiphase HiSafe 3, Ultima-Gold™ AB (biodegradable) and Insta-Gel-XF (non-biodegradable), in terms of [14C]-glucose and [³H]-thymidine counting efficiency. We also analyzed the effect of the relative amount of water (1.6 to 50%), radioisotope concentration (0.1 to 100 nCi/ml), pH (2 to 10) and color of the solutions (samples containing 0.1 to 1.0 mg/ml of Trypan blue) on the counting efficiency in the presence of these scintillation liquids. There were few significant differences in the efficiency of 14C and ³H counting obtained with biodegradable or non-biodegradable scintillation liquids. However, there was an 83 and 94% reduction in the efficiency of 14C and ³H counting, respectively, in samples colored with 1 mg/ml Trypan blue, but not with 0.1 mg/ml, independent of the scintillation liquid used. Considering the low cost of biodegradable scintillation cocktails and their efficacy, these results show that traditional hazardous scintillation fluids may be replaced with the new safe biodegradable fluids without impairment of ³H and 14C counting efficiency. The use of biodegradable scintillation cocktails minimizes both human and environmental exposure to hazardous solvents. In addition, some biodegradable scintillation liquids can be 40% less expensive than the traditional hazardous cocktails.

Pharmacokinetic aspects of Tert-Butylaminoethyl disulfide, an experimental drug against schistosomiasis in mice

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethyl disulfide was performed after administration of two different single doses (35 and 300 mg/kg) of either the cold or labelled drug. Plasma or blood samples were treated with dithiothreitol, perchloric acid, and, after filtration, submitted to further purification with anionic resein. In the final step, the drug was retained on a cationic resin column, eluted with NaCl 1M and detected according to the method of Ellman (1958). Alternatively, radioactive drug was detected by liquid scintillation counting. The results corresponding to the smaller dose of total drug suggested a pharmacokinetic behavior related to a one open compartment model with the following parameters: area under the intravenous curve (AUC i.v.):671 ± 14; AUC oral: 150 ± 40 µg.min. ml [raised to the power of -1]; elimination rate constant: 0.071 min [raised to the power of -1]; biological half life: 9.8 min; distribution volume: 0.74 ml/g. For the higher dose, the results seemed to obey a more complex undertermined model. Combining the results, the occurence of a dose-dependent pharmacokinetic behavior is suggested, the drug being rapidly absorbed and rapidly eliminated; the elimination process being related mainly to metabolization. The drug seems to be more toxic when administered I.V. because by this route it escapes first pass metabolism, while being quickly distributed to tissues. The maximum tolerated blood level seems to be around 16 µg/ml.

Datação radiocarbônica de sítios arqueológicos do tipo sambaqui pela técnica de absorção de CO2: uma alternativa à síntese benzênica

The sambaquis are archaeological sites with remains of pre-historical Brazilian civilizations. They look like small hills containing different kinds of shells, animal and fish bones, small artifacts and even human skeletons. Since the sambaqui sites in the Rio de Janeiro state are younger than 6000 years, the applicability of CO2 absorption on Carbo-Sorb® and 14C determination by counting on a low background liquid scintillation counter was tested. The International Atomic Energy Agency standard reference material IAEA-C2 was used in order to standardize the method. Nine sambaqui samples from five different archaeological sites found in the Rio de Janeiro state were analyzed and 14C ages between 2100 and 3600 years BP were observed. The same samples were sent to the 14C Laboratory of the Centro de Energia Nuclear na Agricultura (CENA/USP) where similar results were obtained.

Neuronal counting and parasympathetic dysfunction in the hearts of chronically Trypanosoma cruzi - infected rats

Ten male Wistar rats, chronically infected with Colombian, São Felipe (12SF) and Y strains of Trypanosoma cruzi and ten non-infected control animals were submitted to the bradycardia responsiveness test, an assessment of heart parasympathetic function, after phenylephrine injection. Six chagasic animals showed heart parasympathetic dysfuntion characterized by reduction in the index of bradycardia baroreflex responsiveness, as compared with the control group. Microscopic examination of the atrial heart ganglia of chagasic rats showed ganglionitis, but no statiscally significant reduction in the number of neurons.

Methodological limitations of counting total leukocytes and thrombocytes in reptiles (Amazon turtle, Podocnemis expansa): an analysis and discussion

The aim of this paper is to compare three different methods for counting white blood cells [WBC] (Natt and Herrick method, estimation with 1,000 and 2,000 erythrocytes) and three methods for counting total thrombocytes [TT] (Wojtaszek method, estimation with 1,000 and 2,000 erythrocytes) in a South American freshwater turtle species, Podocnemis expansa, Schweigger 1812 (Reptilia, Pelomedusidae). Direct WBC counts using the Natt and Herrick method showed limitations, which are discussed here. The WBC and TT counts using 1,000 erythrocytes from blood smears are not recommended for Amazon turtles nor other reptilian species, since wide variation in counts can be observed. Estimation methods for determining WBC and TT based on 2,000 erythrocytes of blood smears were most acceptable because they allow a differentiation between leukocytes and thrombocytes and also had a smaller variation. The methods investigated here for the Amazon turtle, which have been widely used in other reptile species, provided evidence that the most acceptable method is not that of using diluted stains and a hemocytometer.

Evaluation of the PetrifilmTM and TEMPO® systems and the conventional method for counting microorganisms in pasteurized milk

New microbiological methods have been developed and commercialized, but their performance must be guaranteed. The aim of the present study was to evaluate the PetrifilmTM and TEMPO® systems compared to the conventional method for counting microorganisms in pasteurized milk. A total of 141 samples of pasteurized milk were analyzed by counting mesophilic aerobic, Coliforms at 35 ºC, Coliforms at 45 ºC, and Escherichia coli microorganisms. High correlation was found between the methods for counting Coliforms at 35 ºC, but low correlation was found for counting mesophilic aerobic, Coliforms at 45 ºC, and Escherichia coli. No significant statistical difference was found among the three methods for counting Coliforms at 35 ºC; however, the mean counts of mesophilic aerobic, Coliforms at 45 ºC, and Escherichia coli showed significant statistical difference. PetrifilmTM and TEMPO® systems had satisfactory results for Coliforms at 35 ºC in pasteurized milk but low performance for mesophilic aerobic, Coliforms at 45 ºC and Escherichia coli.

Lead, hemoglobin, zinc protoporphyrin and ferritin concentrations in children

OBJECTIVE: To assess the relationship of blood lead and hemoglobin, zinc protoporphyrin, and ferritin concentrations in children. METHODS: A cross-sectional study was carried out in 136 anemic and non-anemic children from two rural villages near a lead smelter in Adrianópolis, Southern Brazil, from July to September 2001. Hemoglobin electrophoresis was performed to exclude children with hemoglobin variants and thalassemia syndromes associated with anemia. Lead was determined by atomic absorption spectrophotometry; hemoglobin by automated cell counting; zinc protoporphyrin by hematofluorometry; ferritin by chemiluminescence. Student's t-test, Mann-Whitney test, and the c² test were used to assess the significance of the differences between the variables investigated in anemic and non-anemic children. Stepwise multivariate linear regression analysis was performed using two models for anemic and non-anemic children respectively. RESULTS: Lead was negatively associated to hemoglobin (p<0.017) in the first model, and in the second model lead was positively associated to zinc protoporphyrin (p<0.004) after controlling for ferritin, age, sex, and per capita income. There was an inverse association between hemoglobin and blood lead in anemic children. It was not possible to confirm if anemic children had iron deficiency anemia or subclinical infection, considering that the majority (90.4%) had normal ferritin. CONCLUSIONS: The study detected a relationship between anemia and elevated blood lead concentrations. Further epidemiological studies are necessary to investigate the impact of iron nutritional interventions as an attempt to decrease blood lead in children.

Labeling of Salmonella Typhymurium with iodine-131 to study phagocytic function in rats

The present study describes a method for labeling Salmonella typhymurium with iodine-131 to evaluate both the morphological and the functional characteristics of the reticulo-endothelial system. A suspension containing 2 x 10(9) bacteria per ml was labeled with carrier-free Na131I without reductor, with a labeling yield of 46.5 ± 3% and 3.5 ± 1.3% of free Iodine-131. The biodistribution of the labeled bacteria in rats was studied with a large field-of-view scintillation camera equiped with a pinhole collimator. Whole body images were obtained 15 and 30 minutes after intravenous injection of the labeled microorganisms. Images showed accumulation of bacteria in the liver and both normal and transplanted spleens of the animals. Autoradiographs of liver and spleen demonstrated labeled bacteria within the cells of the reticulo-endothelial system. The method described is easy to perform, has a good labeling yield and allows the functional evaluation of the reticulo-monophagocytic system, including transplanted spleens.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µ L and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

Influence of variables on centrifuge-flotation technique for recovery of Toxocara canis eggs from soil

The purpose of this study was to evaluate the influence of variables in a flotation technique for the recovery of Toxocara canis eggs from soil. The trials were done under standardized conditions on one gram of previously sterilized soil samples contaminated with 200 eggs of T. canis. The following variables were evaluated in serial steps: sieving; type of wash; time of stirring; resuspension of sediment; solution flotation. Centrifuge-flotation in sodium nitrate (d = 1.20 g/cm³) was adopted as an initial technique, using Tween 80 (0.2%) and decinormal sodium hydroxide as solutions for washing the samples. Ten tests were done to compare the variables, using counting in triplicate. The sieving of the material reduced significantly the recovery of eggs (p < 0.001) and the number of eggs recovered was higher when the sediment was resuspended (p < 0.05). After standardization, flotation solutions sodium chloride, zinc sulfate, sodium dichromate, magnesium sulfate, and sodium nitrate (d = 1.20g/cm³) were compared. The best results were obtained by using zinc sulfate solution. In conclusion, the chances of recovering T. canis eggs from samples using flotation solutions can be increased by washing of soil twice using distilled water, and resuspension of sediment. On the other hand, the sieving procedure can drastically reduce the number of eggs.

HOOKWORM AND THREADWORM INFECTIONS AND THEIR ASSOCIATION WITH HEMOGLOBIN AND EOSINOPHIL CONCENTRATIONS IN RESIDENTS OF SALVADOR-BAHIA, BRAZIL

SUMMARY Parasitic infections are responsible for substantial mortality and morbidity worldwide. In most healthy individuals, little overt pathology is observed during infection with S. stercoralis. However, the symptoms in advanced cases may include gastrointestinal bleeding. Anemia is most commonly associated with hookworm infection, especially when several hundred worms are present. Our study evaluates the relationship between the hookworm or S. stercoralis infection status and the hemoglobin concentration of individuals examined by a private network of laboratories in Salvador, Bahia, Brazil. We examined 374,120 samples from middle-class individuals living in Salvador City from January 2004 to April 2008. The stool samples were analyzed by the Lutz and Baermann-Moraes methods, and the blood samples were analyzed for hemoglobin concentration and eosinophil counting. The prevalence of hookworm and S. stercoralis were 0.27% (1,027) and 0.34% (1,286), respectively. The prevalence of hookworm and S. stercoralis infection was significantly higher in males than in females and increased with age. Eosinophilia was a common laboratorial finding in individuals infected with hookworm and S. stercoralis. The hemoglobin concentration was lower in the hookworm-infected individuals than in non-infected ones, but none of the examined patients were anemic. Lack of anemia could be a consequence of the socioeconomic status of these patients.

FACTORS ASSOCIATED TO ADHERENCE TO DIFFERENT TREATMENT SCHEMES WITH MEGLUMINE ANTIMONIATE IN A CLINICAL TRIAL FOR CUTANEOUS LEISHMANIASIS

The favorable outcome of the treatment of a disease is influenced by the adherence to therapy. Our objective was to assess factors associated with adherence to treatment of patients included in a clinical trial of equivalence between the standard and alternative treatment schemes with meglumine antimoniate (MA) in the treatment of cutaneous leishmaniasis (CL), in the state of Rio de Janeiro. Between 2008 and 2011, 57 patients with CL were interviewed using a questionnaire to collect socioeconomic data. The following methods were used for adherence monitoring: counting of vial surplus, monitoring card, Morisky test and modified Morisky test (without the question regarding the schedule); we observed 82.1% (vial return), 86.0% (monitoring card), 66.7% (Morisky test) and 86.0% (modified Morisky test) adherence. There was a strong correlation between the method of vial counting and the monitoring card and modified Morisky test. A significant association was observed between greater adherence to treatment and low dose of MA, as well as with a lower number of people sleeping in the same room. We recommend the use of the modified Morisky test to assess adherence to treatment of CL with MA, because it is a simple method and with a good performance, when compared to other methods.

Chemotherapeutic effects on larval stages of Schistosoma mansoni during infection and re-infection of mice

The sensitivity of the larval stages of Schistosoma mansoni to chemotherapy with praziquantel and oxamniquine was tested in mice during primary and secondary infections and after different intervals from cercarial exposure. Worm recovery by perfusion of the porto-mesenteric system, followed by counting and a morphometric study of the parasite, allowed the conclusion that the relative resistance of the larval stages of S. mansoni to schistosomicide drugs, demonstrated in primary infections, also persists when the host is already infected. This indicates that a therapeutic failure may result when an infected host is treated some time after being re-infected, because of the presence of migrating, drug-resistant, immature forms of the parasite.

Experimental schistosomiasis in the Common Marmoset Callithrix jacchus

In order to evaluate Callithrix jacchus as an animal model for mansoni schistosomiasis, a group of 10 male animals were once percutaneously exposed to 250 cercariae of the Schistosoma mansoni SLM (São Lourenço da Mata) strain. Animals were periodically bled for measuring serum level of enzymes and proteins and for blood cell counting. When comparing pre-infection to post-infection values, a significant increase was found for alkaline phosphatase at 15 to 120 days p.i., differential counts of eosinophil at 45 and 60 days, and total protein and global eosinophil counts at 120 days. No Schistosoma mansoni eggs were found in stools. Adult worms of small size were recovered from five animals. At day 120, the number of Schistosoma mansoni eggs/g of tissue was 0-289.7 (liver), 0-30.1 (large intestine) and 0-171.4 (small intestine). These findings lead us to classify Callithrix jacchus as a non-permissive host to the SLM strain of Schistosoma mansoni.