Simultaneous circulation of all four dengue serotypes in Manaus, State of Amazonas, Brazil in 2011

INTRODUCTION: Manaus, the capital city of the state of Amazon with nearly 2 million inhabitants, is located in the middle of the Amazon rain forest and has suffered dengue outbreaks since 1998. METHODS: In this study, blood samples were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), aimed at identifying dengue virus serotypes. RESULTS: Acute phase sera from 432 patients were tested for the presence of dengue virus. Out of the 432 patients, 137 (31.3%) were found to be positive. All the four dengue virus serotypes were observed. CONCLUSIONS: The simultaneous circulation of the four dengue serotypes is described for the first time in Manaus and in Brazil.

Extraction and Simultaneous Determination of Glyphosate, AMPA and Compounds of the Shikimic Acid Pathway in Plants

This study has aimed to develop a method for simultaneous extraction and determination by liquid chromatography and mass spectrometry (LC-MS/MS) of glyphosate, aminomethylphosphonic acid (AMPA), shikimic acid, quinic acid, phenylalanine, tyrosine and tryptophan. For the joint analysis of these compounds the best conditions of ionization in mass spectrometry and for chromatographic separation of the compounds were selected. Calibration curves and linearity ranges were also determined for each compound. Different extraction systems of the compounds were tested from plant tissues collected from sugarcane (Saccharum officinarum) and eucalyptus (Eucalyptus urophylla platiphylla) plants two days after the glyphosate application at the dose of 720 g a.e. ha-1. The plant material was dried in a forced air circulation drying oven and in a lyophilizer, and subsequently the extractions with acidified water (pH 2.5), acetonitrile-water (50:50) [v/v] and methanol-water (50:50) [v/v] were tested. To verify the recovery of the compounds in the plant matrix with acidified water as an extracting solution, the samples were fortified with a solution containing the mixture of the different analytical standards present so that this one presented the same levels of 50 and 100 μg L-1 of each compound. All experiments were conducted with three replicates. The analytical method developed was efficient for compounds quantifications. The extraction from the samples dried in an oven and using acidified water allowed better extraction levels for all compounds. The recovery levels of the compounds in the fortified samples with known amounts of each compound for both plants samples were rather satisfactory.

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.

Dispersive liquid-liquid microextraction for the simultaneous separation of trace amounts of zinc and cadmium ions in water samples prior to flame atomic absorption spectrometry determination

In the proposed method, carbon tetrachloride and ethanol were used as extraction and dispersive solvents. Several factors that may be affected on the extraction process, such as extraction solvent, disperser solvent, the volume of extraction and disperser solvent, pH of the aqueous solution and extraction time were optimized. Under the optimal conditions, linearity was maintained between 1.0 ng mL-1 to 1.5 mg mL-1 for zinc and 1.0 ng mL-1 to 0.4 mg mL-1 for cadmium. The proposed method has been applied for determination of trace amount of zinc and cadmium in standard and water samples with satisfactory results.

Simultaneous preconcentration of Cu(II), Cd(II) and Mn(II) on silica-polyethylene glycol and determination by flame atomic absorption spectrometry

A simultaneous solid phase extraction procedure for enrichment of Cu(II), Cd(II) and Mn(II) has been developed. The method is based on adsorption of Cu(II), Cd(II) and Mn(II) ions on polyethylene glycol-silica gel pre-conditioned with acetate buffer (pH 5.5). The adsorbed metal ions are eluted with nitric acid (1 mol L -1) and determined by flame atomic absorption spectrometry. The calibration graph was linear in the range of 2-140 ng mL-1 for Cu(II), 1-40 ng mL-1 for Cd(II) and 4-100 ng mL-1 for Mn(II). The limits of detection were 0.66, 0.33 and 1.20 ng mL-1 for Cu(II), Cd(II) and Mn(II), respectively.

Dispersive liquid-liquid microextraction based on solidification of floating organic drop for simultaneous separation/preconcentration of nickel, cobalt and copper prior to determination by electrothermal atomic absorption spectrometry

A dispersive liquid-liquid microextraction based on solidification of floating organic drop for simultaneous extraction of trace amounts of nickel, cobalt and copper followed by their determination with electrothermal atomic absorption spectrometry was developed. 300 µL of acetone and 1-undecanol was injected into an aqueous sample containing diethyldithiocarbamate complexes of metal ions. For a sample volume of 10 mL, enrichment factors of 277, 270 and 300 and detection limits of 1.2, 1.1 and 1 ng L-1 for nickel, cobalt and copper were obtained, respectively. The method was applied to the extraction and determination of these metals in different water samples.

Simultaneous analysis of saturated and unsaturated fatty acids present in pequi fruits by capillary electrophoresis

In the current study, an alternative method has been proposed for simultaneous analysis of palmitic, stearic, oleic, linoleic, and linolenic acids by capillary zone electrophoresis (CZE) using indirect detection. The background electrolyte (BGE) used for the analysis of these fatty acids (FAs) consisted of 15.0 mmol L−1 NaH2PO4/Na2HPO4 at pH 6.86, 4.0 mmol L−1 SDBS, 8.3 mmol L−1 Brij 35, 45% v/v acetonitrile (can), and 2.1% n-octanol. The FAs quantification of FAs was performed using a response factor approach, which provided a high analytical throughput for the real sample. The CZE method, which was applied successfully for the analysis of pequi pulp, has advantages such as short analysis time, absence of lipid fraction extraction and derivatization steps, and no significant difference in the 95% confidence intervals for FA quantification results, compared to the gas chromatography official method (AOCS Ce 1h-05).

Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.

Simultaneous quantitation of serum HBV DNA and HBeAg can distinguish between slow and fast viral responses to antiviral therapy in patients with chronic hepatitis B

BACKGROUND: The quantitation of serum HBeAg is not commonly used to monitor viral response to therapy in chronic hepatitis B. METHODS: In this study, 21 patients receiving varying therapies were followed and their viral response monitored by concomitant viral load and HBeAg quantitation in order to study the meaning and the kinetics of both parameters. RESULTS: It was possible to distinguish between three different patterns of viral response. The first was characterized by a simultaneous decrease in serum HBV DNA and HBeAg. The second pattern was characterized by a decrease in serum HBeAg but persistent detection of HBV DNA. The third pattern was characterized by undetectable HBV DNA with persistent HBeAg positivity, which points to a non-response (Pattern III-B) except when HBeAg levels showed a slow but steady drop, characterizing a "slow responder" patient (Pattern III-A). CONCLUSIONS: The first pattern is compatible with a viral response. A long-term HBeAg seropositivity with a slow and persistent decrease (Pattern III-A) is also compatible with a viral response and calls for a prolongation of anti-viral treatment.

High prevalence of the simultaneous excretion of polyomaviruses JC and BK in the urine of HIV-infected patients without neurological symptoms in São Paulo, Brazil

OBJECTIVE: To evaluate the prevalence of the urinary excretion of BKV and JCV in HIV-infected patients without neurological symptoms. METHODS: Urine samples from HIV-infected patients without neurological symptoms were tested for JC virus and BK virus by PCR. Samples were screened for the presence of polyomavirus with sets of primers complementary to the early region of JCV and BKV genome (AgT). The presence of JC virus or BK virus were confirmed by two other PCR assays using sets of primers complementary to the VP1 gene of each virus. Analysis of the data was performed by the Kruskal-Wallis test for numerical data and Pearson or Yates for categorical variables. RESULTS: A total of 75 patients were included in the study. The overall prevalence of polyomavirus DNA urinary shedding was 67/75 (89.3%). Only BKV DNA was detected in 14/75 (18.7%) urine samples, and only JCV DNA was detected in 11/75 (14.7%) samples. Both BKV and JCV DNA were present in 42/75 (56.0%) samples. CONCLUSION: In this study we found high rates of excretion of JCV, BKV, and simultaneous excretion in HIV+ patients. Also these results differ from the others available on the literature.

Simultaneous occurrence of pulmonary tuberculosis and carcinomatous lymphangitis

Tuberculosis is an important cause of mortality due to its high prevalence, considering that one third of the world’s population is infected with the tuberculosis bacillus. We report the first case of carcinomatous lymphangitis associated with active pulmonary tuberculosis. Carcinomatous lymphangitis is a rare event that may be confounded with tuberculosis because of its radiographic and clinical characteristics.

Simultaneous occurrence of brain tumor and myeloradiculopathy in schistosomiasis mansoni: case report

Simultaneous occurrence of brain tumor and myeloradiculopathy in cases of Manson's schistosomiasis have only rarely been described. We report the case of a 38-year-old man who developed seizures during a trip to Puerto Rico and in whom a brain tumor was diagnosed by magnetic resonance imaging: brain biopsy revealed the diagnosis of schistosomiasis. He was transferred to a hospital in the United States and, during hospitalization, he developed sudden paraplegia. The diagnosis of myeloradiculopathy was confirmed at that time. He was administered praziquantel and steroids. The brain tumor disappeared, but the patient was left with paraplegia and fecal and urinary dysfunction. He has now been followed up in Brazil for one year, and his clinical state, imaging examinations and laboratory tests are presented here.

Arboviral diseases in the Western Brazilian Amazon: a perspective and analysis from a tertiary health & research center in Manaus, State of Amazonas

The Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), located in Manaus, the capital of the State of Amazonas (Western Brazilian Amazon), is a pioneering institution in this region regarding the syndromic surveillance of acute febrile illness, including arboviral infections. Based on the data from patients at the FMT-HVD, we have detected recurrent outbreaks in Manaus by the four dengue serotypes in the past 15 years, with increasing severity of the disease. This endemicity has culminated in the simultaneous circulation of all four serotypes in 2011, the first time this has been reported in Brazil. Between 1996 and 2009, 42 cases of yellow fever (YF) were registered in the State of Amazonas, and 71.4% (30/42) were fatal. Since 2010, no cases have been reported. Because the introduction of the yellow fever virus into a large city such as Manaus, which is widely infested by Aedes mosquitoes, may pose a real risk of a yellow fever outbreak, efforts to maintain an appropriate immunization policy for the populace are critical. Manaus has also suffered silent outbreaks of Mayaro and Oropouche fevers lately, most of which were misdiagnosed as dengue fever. The tropical conditions of the State of Amazonas favor the existence of other arboviruses capable of producing human disease. Under this real threat, represented by at least 4 arboviruses producing human infections in Manaus and in other neighboring countries, it is important to develop an efficient public health surveillance strategy, including laboratories that are able to make proper diagnoses of arboviruses.