Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Laser assisted extraction of pacemaker and implantable defibrillator leads

OBJECTIVE: To analyze the results of laser-assisted extraction of permanent pacemaker and defibrillator leads. METHODS: We operated upon 36 patients, whose mean age was 54.2 years, and extracted 56 leads. The reasons for extracting the leads were as follows: infection in 19 patients, elective replacement in 13, and other causes in 4 patients. The mean time of catheter placement was 7.5±5.5 years. Forty-seven leads were from pacemakers, and 9 were from defibrillators. Thirty-eight leads were in use, 14 had been abandoned in the pacemaker pocket, and 4 had been abandoned inside the venous system. RESULTS: We successfully extracted 54 catheters, obtaining a 96.4% rate of success and an 82.1% rate for complete extraction. The 2 unsuccessful cases were due to the presence of calcium in the trajectory of the lead. The mean duration of laser light application was 123.0±104.5 s, using 5,215.2±4,924.0 pulses, in a total of 24.4±24.2 cycles of application. Thirty-four leads were extracted from the myocardium with countertraction after complete progression of the laser sheath, 12 leads came loose during the progression of the laser sheath, and the remaining 10 were extracted with other maneuvers. One patient experienced cardiac tamponade after extraction of the defibrillator lead, requiring open emergency surgery. CONCLUSION: The use of the excimer laser allowed extraction of the leads with a 96% rate of success; it was not effective in 2 patients who had calcification on the lead. One patient (2.8%) had a complication that required cardiac surgery on an emergency basis.

Schistosoma mansoni antigenic extracts obtained by different extraction procedures

Solubilization of Schistosoma mansoni antigens was obtained by agitation of adult worms in a 3M KCl solution. The protein contents of the KCl extrats varied from 0.35 to 0.96 mg/ml. Sera from 97 patients with hepatointestinal shistosomiasis and viable eggs in stools from a Brazilian endemic area were studied by immunoelectroomophoresis and Ouchterlony immunodiffusion methods with the KCl extract and with another antigen, obtained by homogenization of adult schistosomes in saline. The rate of positiveness of immunoprecipitation deterctions by immunoelectroomophoresis with the KCl extract was 53.5%. A correlation was verified between methods of detection and extration procedures, resulting in a better association of the extract obtained by agitation in 3M KCl and immunoelectroomophoresis.

Effect of magnesium chloride and guanidinum chloride on the extraction of components of extracellular matrix from chicken cartilage

In order to evaluate the effect of chaotropic agents on proteoglycan and non-collagenous proteins, chicken xiphoid cartilage was treated with guanidine-HCI and MgCl2 in different concentrations (1M to 5M), and different periods of time (12, 24, 48 and 72hr). The maximum yield of uronic acid was obtained with 3M MgCl2 (73.3 per cent). Concentrations of 4M and 5M of MgCl2 showed that much less uronic acid was removed, 55.3 per cent and 38.1 respectively. Extraction with 3M MgCl2 and 3M guanidine-HCl resulted better efficiency when performed for 48 hr. Analysis by SDS-PAGE of the extracts obtained with guanidine-HCl and MgCl, in different concentrations pointed out that most components are equally removed with the two solvents, showing that the extraction with MgCl2 is an alternative assay to remove non-collagenous proteins from extracellular matrix.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin (Btmed) was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200). Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins.

Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by enzyme-linked immunosorbent assay

The present study was conducted to detected IgG antibodies using Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by the enzyme-linked immunosorbent assay (ELISA). Sera from 90 subjects were analyzed (30 with strongyloidiasis, 30 with other parasites and 30 healthy individuals). Results were expressed in antibody titers, which were considered as positive when titer was > 80. Sensibility and specificity of the assay were 100% and 96.7%, respectively. It can be concluded that the heterologous alkaline extract could be employed in ELISA as a diagnostic aid in human strongyloidiasis, due to its advantages as easiness of obtaining, practicability in preparing, and high indexes of sensitivity and specificity.

Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni stages

Schistosomes are endoparasites causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand the schistosomiasis epidemiology and disease transmission patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction method of egg, larval and adult stages of Schistosoma mansoni. One euro makes possible to perform 60,000 DNA extraction reactions at top speed (only 15 min of incubation and 5 handling steps).

Night and crepuscular mosquitoes and risk of vector-borne diseases in areas of piassaba extraction in the middle Negro River basin, state of Amazonas, Brazil

A study of crepuscular and night-biting mosquitoes was conducted at remote settlements along the Padauiri River, middle Negro River, state of Amazonas, Brazil. Collections were performed with human bait and a CDC-light trap on three consecutive days per month from June 2003-May 2004. In total, 1,203 h of collection were performed, of which 384 were outside and 819 were inside houses. At total of 11,612 specimens were captured, and Anophelinae (6.01%) were much less frequent than Culicinae (93.94%). Anopheles darlingi was the most frequent Anophelinae collected. Among the culicines, 2,666 Culex (Ae.) clastrieri Casal & Garcia, 2,394 Culex. (Mel.) vomerifer Komp, and 1,252 Culex (Mel.) eastor Dyar were the most frequent species collected. The diversity of insects found reveals the receptivity of the area towards a variety of diseases facilitated by the presence of vectors involved in the transmission of Plasmodium, arboviruses and other infectious agents.

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods

The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20% of the AdV, 90% of the RV and 100% of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8% of the AdV and 90% of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.

Evaluating leaf litter beetle data sampled by Winkler extraction from Atlantic forest sites in southern Brazil

Evaluating leaf litter beetle data sampled by Winkler extraction from Atlantic forest sites in southern Brazil. To evaluate the reliability of data obtained by Winkler extraction in Atlantic forest sites in southern Brazil, we studied litter beetle assemblages in secondary forests (5 to 55 years after abandonment) and old-growth forests at two seasonally different points in time. For all regeneration stages, species density and abundance were lower in April compared to August; but, assemblage composition of the corresponding forest stages was similar in both months. We suggest that sampling of small litter inhabiting beetles at different points in time using the Winkler technique reveals identical ecological patterns, which are more likely to be influenced by sample incompleteness than by differences in their assemblage composition. A strong relationship between litter quantity and beetle occurrences indicates the importance of this variable for the temporal species density pattern. Additionally, the sampled beetle material was compared with beetle data obtained with pitfall traps in one old-growth forest. Over 60% of the focal species captured with pitfall traps were also sampled by Winkler extraction in different forest stages. Few beetles with a body size too large to be sampled by Winkler extraction were only sampled with pitfall traps. This indicates that the local litter beetle fauna is dominated by small species. Hence, being aware of the exclusion of large beetles and beetle species occurring during the wet season, the Winkler method reveals a reliable picture of the local leaf litter beetle community.

Extraction of soil water by plants: development and validation of a model

A quantitative model of water movement within the immediate vicinity of an individual root is developed and results of an experiment to validate the model are presented. The model is based on the assumption that the amount of water transpired by a plant in a certain period is replaced by an equal volume entering its root system during the same time. The model is based on the Darcy-Buckingham equation to calculate the soil water matric potential at any distance from a plant root as a function of parameters related to crop, soil and atmospheric conditions. The model output is compared against measurements of soil water depletion by rice roots monitored using γ-beam attenuation in a greenhouse of the Escola Superior de Agricultura "Luiz de Queiroz"/Universidade de São Paulo(ESALQ/USP) in Piracicaba, State of São Paulo, Brazil, in 1993. The experimental results are in agreement with the output from the model. Model simulations show that a single plant root is able to withdraw water from more than 0.1 m away within a few days. We therefore can assume that root distribution is a less important factor for soil water extraction efficiency.

Extraction and concentration of biogenic calcium oxalate from plant leaves

The objective of this study was to extract and concentrate calcium oxalate (CaOx) crystals from plant leaves that form the above mentioned crystals. The chemical and physical studies of CaOx from plant to be performed depend on an adequate amount of the crystals. The plant used in this study was croton (Codiaeum variegatum). The leaves were ground in a heavy duty blender and sieved through a 0.20 mm sieve. The suspension obtained was suspended in distilled water. The crystals were concentrated at the bottom of a test tube. The supernatant must be washed until it is free of plant pigments and other organic substances. Biogenic CaOx crystals have well-defined and sharp peaks, indicating very high crystallinity. Moreover, the CaOx crystals were not damaged during the extraction procedure, as can be seen on the scanning electron microscope images. The porposed method can be considered efficient to extract and concentrate biogenic calcium oxalate.

Comparison of H/Al stoichiometry of mineral and organic soils in Brazil

Exchangeable Al has been used as a criterion for the calculation of lime requirement in several Brazilian States. However, the laboratory method with extraction by a 1 mol L-1 KCl solution followed by indirect alkaline titration is not accurate for some Brazilian soils, mainly in the case of soils with high organic matter content. The objective of this study was therefore to evaluate the stoichiometry of H+/Al3+ in KCl soil extracts. The results suggested that organically complexed Al is the main contributor to exchangeable acidity in soils enriched with organic matter. Liming recommendations for organic soils based exclusively on exchangeable Al determined by the NaOH titration method should therefore be revised.