Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.

Fecal specimens preparation methods for PCR diagnosis of human taeniosis

Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques

The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage) it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.

Serological, clinical and epidemiological evaluation of toxocariasis in urban areas of south Brazil

Toxocariasis is a worldwide public-health problem that poses major risks to children who may accidentally ingest embryonated eggs of Toxocara. The objectives of this study were to investigate the occurrence of anti-Toxocara spp. antibodies in children and adolescents and the variables that may be involved, as well as environmental contamination by Toxocara spp. eggs, in urban recreation areas of north central mesoregion, Paraná State, Brazil. From June 2005 to March 2007. a total of 376 blood samples were collected by the Public Health Service from children and adolescents one to 12 years old, of both genders. Samples were analyzed by the indirect ELISA method for detection of anti-Toxocara antibodies. Serum samples were previously absorbed with Ascaris suum antigens, and considered positive with a reagent reactivity index >1. Soil samples from all of the public squares and schools located in the four evaluated municipalities that had sand surfaces (n = 19) or lawns (n = 15) were analyzed. Of the 376 serum samples, 194 (51.6%) were positive. The seroprevalence rate was substantially higher among children aging one to five years (p = 0.001) and six to eight years (p = 0.022). The clinical signs and symptoms investigated did not show a statistical difference between seropositive and seronegative individuals (p > 0.05). In 76.5% of the investigated recreation places, eggs of Toxocara were detected in at least one of the five collected samples. Recreation areas from public schools were 2.8 times more contaminated than from public squares. It is important to institute educational programs to inform families and educators, as well as to improve sanitary control of animals and cleaning of the areas intended for recreation in order to prevent toxocariasis.

Syndromic surveillance: etiologic study of acute febrile illness in dengue suspicious cases with negative serology. Brazil, Federal District, 2008

With the aim of identifying the etiology of acute febrile illness in patients suspected of having dengue, yet with non reagent serum, a descriptive study was conducted with 144 people using secondary serum samples collected during convalescence. The study was conducted between January and May of 2008. All the exams were re-tested for dengue, which was confirmed in 11.8% (n = 17); the samples that remained negative for dengue (n = 127) were tested for rubella, with 3.9% (n = 5) positive results. Among those non reactive for rubella (n = 122), tests were made for leptospirosis and hantavirus. Positive tests for leptospirosis were 13.9% (n = 17) and none for hantavirus. Non reactive results (70.8%) were considered as Indefinite Febrile Illness (IFI). Low schooling was statistically associated with dengue, rubella and leptospirosis (p = 0.009), dyspnea was statistically associated with dengue and leptospirosis (p = 0.012), and exanthem/petechia with dengue and rubella (p = 0.001). Among those with leptospirosis, activities in empty or vacant lots showed statistical association with the disease (p = 0.013). Syndromic surveillance was shown to be an important tool in the etiologic identification of IFI in the Federal District of Brazil.

THE FIRST CASE OF Angiostrongylus cantonensis EOSINOPHILIC MENINGITIS DIAGNOSED IN THE CITY OF SÃO PAULO, BRAZIL

Introduction: Angiostrongylus cantonensis is a natural parasite found in lung arteries of rats, which in humans may cause eosinophilic meningitis. Objective: To report the first case of eosinophilic meningitis caused by Angiostrongylus cantonensis in the city of São Paulo, Brazil. Case report: A male patient, 11 years old, living in the southern area of São Paulo, was admitted to the Pediatric Emergency Department with ongoing headaches for three days, but no fever or any other complaint. The presence of snails and rodents was reported in the peridomicile. The child was awake, lucid, oriented; muscular strength preserved, isochoric, photo reagent pupils and terminal nuchal rigidity - Glasgow Coma Scale (GCS) = 15. The laboratory tests showed a mild leukocytosis with 1736 eosinophils/mm3 and the CSF analysis disclosed 160 leukocytes/mm3 with 36% of eosinophils. The bacterial culture was negative. Computed Cerebral Tomography showed no alterations. The RT-PCR assay for detecting Angiostrongylus cantonensis larvae and DNA was negative. ELISA antibodies for IgG anti-A. cantonensis was negative in serum and undetermined in CSF and samples collected five days after the onset of symptoms. Seroconversion was observed in the sample collected 135 days later. Conclusion: the epidemiological and clinical data, the CSF alterations with eosinophilia and the seroconversion strongly suggest Angiostrongylus cantonensis eosinophilic meningitis.

SEROPREVALENCE OF CHAGASIC INFECTION IN YOUNG INDIVIDUALS IN A BLOOD CENTER IN THE STATE OF SAO PAULO, BRAZIL

SUMMARY This study aimed at estimating the number of cases of non-negative serological reactions to Chagas disease in blood donors at the Blood Center of Botucatu, São Paulo, Brazil, from 2003 to 2010 and at relating them to their cities of origin. Five hundred and seventy-four non-negative results for Chagas disease were evaluated. Of these, 371 (64.8%) were reagent, and 203 (35.4%) were inconclusive. The prevalence of Chagas disease in blood donors was 0.05%. There were, on average, 72 cases/year, and a prevalence of males was observed (64.8%). Forty-three (7.49%) individuals were 18 to 30 years old; 92 (16.02%) were 31 to 40; 147 (25.61%) 41 to 50, and 292 (50.87%) were older than 50 years. It was observed that 29.3% of females with reagent serology were at their fertile age (18 and 45 years). The majority of donors were originally from cities in the southwestern and central regions of São Paulo, but individuals from other states contributed with 20%. The provenance of most donors was the city of Botucatu/SP, followed by the city of Taquarituba/SP. Therefore, the profile of donors at this blood center favors the occurrence of a larger number of non-negative serological reactions. Although there has been a significant reduction in the number of new cases/year for this disease, it is still a public-health problem, and results suggest the need for new epidemiological assessments in the studied region.

Overexpressing BDNF in Neural Stem Cells using non-viral gene delivery strategies

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Evaluation of preventative and control measures for congenital syphilis in State of Mato Grosso

Introduction Congenital syphilis is an important health problem in Brazil. This study assessed measures aimed at the prevention and control of syphilis in the State of Mato Grosso and its capital, Cuiabá. Methods A descriptive study cross-sectional and of time trends assessing the congenital syphilis was performed in Cuiabá and Mato Grosso between 2001 and 2011. We compared maternal sociodemographic characteristics and health care utilization related to cases of congenital syphilis during the periods from 2001 to 2006 and from 2007 to 2011. We assessed the temporal trends in this disease's incidence using a simple linear regression. Results Between 2001 and 2006 in Mato Grosso, 86.8% of the mothers who had live births with congenital syphilis received prenatal care, 90.6% presented with a nontreponemal test reagent at delivery, 96.2% had no information regarding a treponemal confirmatory test at delivery, and 77.6% received inadequate treatment for syphilis; additionally, 75.8% of their partners were not treated. There was a statistically significant reduction in prenatal visits (p = 0.004) and an increase in the proportion of mothers reactive to nontreponemal tests at delivery (p = 0.031) between the two periods. No other variables were found to differ significantly between the periods. In Cuiabá, we observed a similar distribution of variables. In the state and in the capital, the increasing trend of congenital syphilis was not statistically significant. Conclusions The high incidence of congenital syphilis in Mato Grosso and the low levels of health care indicators for pregnant women with syphilis suggest the need to improve the coverage and quality of prenatal care.

Photolabile protection for amino acids: studies on the release from novel benzoquinolone cages

The synthesis of a novel fused nitrogen heterocycle, benzoquinolone, for evaluation as a photocleavable protecting group is described for the first time, by coupling to model amino acids (alanine, phenylalanine and glutamic acid). Conversion of the phenylalanine ester conjugate to the thionated derivative was accomplished by reaction with Lawesson’s reagent. Photocleavage studies of the carbonyl and thiocarbonyl benzoquinolone conjugates in various solvents and at different wavelengths (300, 350 and 419 nm) showed that the most interesting result was obtained at 419 nm for the thioconjugate, revealing that the presence of the thiocarbonyl group clearly improved the photolysis rates, giving practicable irradiations times for the release of the amino acids (less than 1 minute).

Microfluidic based on aqueous two-phase system for plasmidic DNA purification

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biotecnologia)

Sôbre a determinação do nitrogênio, do fósforo e do potássio no mesmo extrato

The determination of total nitrogen, phosphorus, and potassium in plant material can be carried out in a common extract prepared with sulphuric acid and 30 per cent hydrogen peroxide. Nitrogen is estimated by direct nesslerization of a suitable aliquot (1-5 ml of the 50 ml extract made out of 250 mg of dried material); in order to avoid excessive acidity, 10 ml of Nessler's reagent should be employed. An aliquot of 1-5 ml suffices for the colorimetric determination of phosphorus by the molybdenum method; to reduce the phosphomolybdate complex 2 ml of a 2% SnC12 soln are necessary. Potassium is determined by the cobaltinitrite method after elimination of ammonium salts with the aid of aqua-regia.

Estudos sôbre óleos do fígado de cacão Desmobrânquios da família Sphyrnidae, Mir. Rib., gêneros Carcharias, Raf., Galeocerdo, Ranz., e Odontaspis, Shau

1. The author suggests a tecnique for the determination of vitamin A on shark liver oils in industrial plants. The advantages of using oly four ml. of reagent and of permitting a quickly rigorous reading by photoeletric cell, contribute to the possibility of the examination of a great number of samples daily; 2. It is described a survey on the vitamin A content of oils from shark livers, which has been made at the Finishing School Darcy Vargas, Marammaia Is., Rio de Janeiro State. The conclusions are the following: a) Male individuals have showed generally tendency for higher vitamin A pontency oils; b) The size of the fish does not interfere in the vitamin content of the oil (graphic 4); c) The data collected upon 3.085 individuals led to the conclusion that some species are richer in the reservated vitamin although it was possible to catch in the same specie fishes with widely variable potency in vitamin A. One fish belonging to the specie C. lamia produced the highest vitamin potency oil with 167.712 international units per gram; d) The fishing season appears to have no influence on the oils; e) The adventitous food seems to be the most important factor affecting the content of vitamin A of the shark-liver oils; 3. The presence and the quantity of vitamin D in those oils was investigated and two of the determinations are presented.

Emprêgo do teste químico para o estudo da eliminação das gonadotropinas durante o ciclo menstrual

A chemical test previously described for the diagnosis of pregnancy was applied to the study of the excretion of gonadotropin in the urine during menstrual cycle. The chemical test is based on the selective adsorption by kaolim of the reducing substances biologically related to urinary gonadotropin. The active substance when acidified to pH 4.0 is adsorbed by the kolin and eluated with O.1N sodium hydroxide. The alkaline solution is treated by Somogyi's copper reagent and the excess not reduced is titrated by 0.005 N sodium thiosulfate. Gonadotropin is quantitatively addorbed by kaolin at pH 4.0 and eluated by alkaline solution as previously demonstrated by the A. (1). In the present paper the complete menstrual cycle was studied daily. It was observed that normally there are two distinct maxima of excretion. This study is based on 11 normal cycles (24-30 days) and 34 abnormal ones. Normal cycles showed a intramenstrual estrogens elimination from 200 to 260 mice units determinated by the Allen - Doisy full estrus smear test. The abnormal cycles belonging also to normal women showed much less estrogen excretion (14 to 25 mice units) Table II). In those cases with decreased estrogen excretion no fall in the curve after 14 th. day was observed. The A. suggest that the peaks of gonadotropin excretion is not related to the oculation but possibly due, the first one, to the follicle stimulating hormone and the second to the luteinizing hormone of hormone stimulating of the inerstitial tissue.