Elétrodo de pasta de carbono molecularmente impresso para deteção voltamétrica de Furazolidona

A furazolidona é uma substância ativa do medicamento Giarlam que contém um espetro anti-bacteriano relativamente amplo e que é frequentemente usado para tratar certas doenças bacterianas e protozoárias no homem. A maioria dos fármacos exige uma dosagem que garanta os níveis de segurança e eficácia de atuação. A necessidade de dosear os medicamentos e os seus metabólitos exige o desenvolvimento constante de métodos analíticos eficientes. Neste trabalho desenvolveu-se um novo sensor eletroquímico para a deteção da furazolidona, baseado num elétrodo de pasta de carbono modificado com um polímero molecularmente impresso. A procura de novos materiais que permitam uma melhor seletividade e sensibilidade aos sistemas de deteção é especialmente importante no desenvolvimento de métodos analíticos. Os polímeros molecularmente impressos enquadram-se nesse perfil e o seu uso tem vindo a ser cada vez mais frequente como ferramenta importante em química analítica. Assim, sintetizou-se um polímero com cavidades seletivas para a Furazolidona. Este polímero foi, misturado com grafite e perafina de modo a produzir uma pasta de carbono. Uma seringa de plástico foi usada como suporte da pasta de carbono. O comportamento eletroquímico do sensor foi avaliado e diversas condições de utilização foram estudadas e otimizadas. O sensor apresenta um comportamento linear entre a intensidade do pico e a concentração numa gama de concentrações entre 1 e 100 μM, um limite de deteção de 1 μM e uma precisão (repetibilidade) inferior a 7%. A aplicabilidade do sensor fabricado em amostras complexas foi avaliada pela deteção do fármaco em amostras de urina.

Molecularly imprinted sensor for voltammetric detection of norfloxacin

In this work, a norfloxacin selective modified glassy carbon electrode (GCE) based on a molecularly imprinted polymer (MIP) as electrochemical sensor was developed. A suspension of multi-walled carbon nanotubes (MWCNTs) was deposited on the electrode surface. Subsequently, a molecularly imprinted film was prepared by electropolymerization, via cyclic voltammetry of pyrrole (PPy) in the presence of norfloxacin (NFX) as the template molecule. A control electrode (NIP) was also prepared. Scanning electron microscopy (SEM) and cyclic voltammetry in a ferrocyanide solution were performed for morphological and electrochemical characterisation, respectively. Several experimental parameters were studied and optimised. For quantification purposes the MIP/MWCNT/GCE was immersed in NFX solutions for 10 min, and the detection was performed in voltammetric cell by square wave voltammetry. The proposed sensor presented a linear behaviour, between peak current intensity and logarithmic concentration of NFX between 1 × 10−7 and 8 × 10−6 M. The obtained results presented good precision, with a repeatability of 4.3% and reproducibility of 9% and the detection limit was 4.6 × 10−8 M (S/N = 3). The developed sensor displayed good selectivity and operational lifetime, is simple to fabricate and easy to operate and was successfully applied to the analysis of NFX in urine samples.

Candiduria in a public hospital of São Paulo (1999-2004): characteristics of the yeast isolates

The study involved 100 yeast isolates, obtained from urine samples provided by a Public Pediatric Hospital of São Paulo, Brazil, from 1999 to 2004. The most frequent species was Candida albicans, followed by C. tropicalis, C. glabrata and C. parapsilosis. In regard to virulence, 97% of the isolates showed index 3 for proteinase and 63% index 2 for phospholipase. The most frequent killer biotypes were 511 and 888.

Primary Disorders of Neurotransmitter Metabolism: Experience of a Tertiary Center

Neurotransmitter diseases are a group of inherited disorders attributable to a disturbance of neurotransmitter metabolism. Biogenic amines are neurotransmitters with multiple roles including psychomotor function, hormone secretion, cardiovascular, respiratory and gastrointestinal control, sleep mechanisms, body temperature and pain. Given the multiple functions of monoamines, disorders of their metabolism comprise a wide spectrum of manifestations, with motor dysfunction being the most prominent clinical feature. Methods: Case review of 12 patients from 4 families, with primary disorders of biogenic amine metabolism. Results: Aromatic L-amino acid decarboxylase deficiency (4 patients from 2 families), and GTP-cyclohydrolase (8 patients from 2 families) were the two diseases identified. Age at first symptoms varied between 2 months and 6 years. Developmental delay was present in all cases except 2 patients with GTP cyclohydrolase deficiency. The combination of axial hypotonia and limb dystonia was also frequent. Children with aromatic L-amino acid decarboxylase deficiency exhibited temperature instability, oculogyric crisis and disturbances of sleep. The index case of one family with GTP cyclohydrolase deficiency presented with Parkinsonism (bradykinesia, rigidity and hypomimia). Analysis of neurotransmitters and their metabolites in CSF was crucial for the identification of index cases. Response to therapy was variable but in general unsatisfactory except in a family with GTP cyclohydrolase deficiency. Conclusions: These disorders should be considered in the differential diagnosis of paediatric neurodegenerative diseases, in order to allow an adequate therapeutic trial that can favor prognosis.

Transmissão do vírus citomegálico através do aleitamento materno em prematuros

RESUMO: Nas últimas quatro décadas, desenvolveram-se vários estudos, dispersos por todo o mundo, visando analisar a importância da transmissão perinatal do CMV pelo leite materno, nos recém-nascidos prematuros e de baixo peso à nascença. Comparando estes estudos, as taxas de incidência de infecção e doença são extremamente variáveis, não havendo consenso entre os autores. Surgiu, assim, a necessidade de realizar a presente dissertação, pioneira ao nível nacional, com o objectivo de determinar a incidência da infecção perinatal citomegálica, transmitida através do aleitamento materno a recémnascidos prematuros, e identificar as suas consequências clínicas. Para a elaboração deste trabalho foram seleccionados todos os recém-nascidos com idade gestacional inferior a 35 semanas e respectivas mães seropositivas para CMV, internados na Unidade de Cuidados Intensivos de Neonatalogia do Hospital de São Francisco Xavier, entre o período de 1 de Outubro de 2010 e 31 Julho de 2011. Foram analisadas as urinas dos recém-nascidos e o leite materno das mães na primeira, sexta e décima segunda semana pós-parto, utilizando a técnica de Nested-PCR e, posteriormente, a PCR em Tempo Real, para determinar a carga viral do leite materno. Constatou-se que a aquisição da infecção perinatal de CMV no recém-nascido prematuro ocorre com alguma frequência (40,5%), sendo muito provável que a via de transmissão em 38,1% destes casos seja o leite materno infectado, não devendo esta ser desvalorizada. Relativamente às consequências clínicas, não foram observadas alterações clínico-laboratoriais associadas à infecção citomegálica. Analisando os factores que condicionam a transmissão da infecção citomegálica perinatal, estabeleceu-se uma correlação entre a carga viral do leite materno e a transmissão do vírus, permitindo deduzir que quanto maior a carga viral, maior o risco de transmissão. Finalmente, os resultados obtidos sugerem que o risco de transmissão da infecção e suas consequências clínicas não justificam a contra-indicação da amamentação, pois esta comporta, também, elevados benefícios. ----------------ABSTRACT: In the last four decades, several studies were developed all over the world to analyze the importance of CMV perinatal transmission through mother´s milk in the preterm and low birthweight newborns. Comparing these studies, the infection and disease incidence rates are extremely variable, without agreement between the authors. The aim of this study, the first of the kind made in Portugal, is to define the rate of perinatal cytomegalovirus infection in preterm newborns via breastfeeding and its clinical consequences. All the newborns with gestational age below 35 weeks and their CMV seropositive mothers, admitted between the October 1, 2010 and July 31, 2011 in the Neonatology Intensive Care Unit of the São Francisco Xavier Hospital, were included in this study. Human breast milk and urine specimens were collected from mothers and their preterms infants around the 1st, 6th and 12th week after delivery and analyzed by Nested-PCR. The PCR in Real Time was used to determine the breast milk viral load. It was found that the CMV perinatal infection in preterm infants occurs in 40,5% of the cases and most likely 38,1% of these were infected via breastmilk and therefore this should not be overlooked. Considering the clinical consequences, no clinical and laboratory changes associated with cytomegalovirus infection were observed. Analyzing the factors that determine the perinatal transmission of the cytomegalovirus, it was established a correlation between breast milk viral load and viral transmission, allowing to conclude that the higher the viral load the greater the risk of transmission. Finally, the obtained results suggest that the risk of CMV infection and its clinical consequences don´t justify the breastfeeding contraindication because it also provides high benefits.

Hyponatremia in visceral leishmaniasis

There are few reports linking hyponatremia and visceral leishmaniasis (kala-azar). This is a study of 55 consecutive kala-azar patients and 20 normal individuals as a control group. Hyponatremia and serum hypo-osmolality were detected in 100% of kala-azar patients. High first morning urine osmolality (750.0 ± 52.0 vs. 894.5 ± 30.0mOsm/kg H2O, p < 0.05), and high 24-hour urine osmolality (426.0 ± 167.0 vs. 514.6 ± 132.0 mOsm/kg H2O, p < 0.05) demonstrated persistent antidiuretic hormone secretion. Urinary sodium was high (82.3 ± 44.2 vs.110.3 ± 34.7 mEq/L, p < 0.05). Low seric uric acid occurred in 61.8% of patients and increased fractional urinary uric acid excretion was detected in 74.5% of them. Increased glomerular filtration rate was present in 25.4% of patients. There was no evidence of extracellular volume depletion. Normal plasma ADH levels were observed in kala-azar patients. No endocrine or renal dysfunction was detected. It is possible that most hyponatremic kala-azar patients present the syndrome of inappropriate antidiuretic hormone secretion.

Leflunomide and Polyomavirus-Associated Nephropathy in Renal Transplant

Polyomavirus nephropathy is a major complication in renal transplantation, associated with renal allograft loss in 14 to 80% of cases. There is no established treatment, although improvement has been reported with a variety of approaches. The authors report two cases of polyomavirus infection in renal allograft recipients. In the first case, a stable patient presented with deterioration of renal function, worsening hypertension and weight gain following removal of ureteral stent placed routinely at the time of surgery. Ultrasound examination and radiology studies revealed hydronephrosis due to ureteral stenosis. A new ureteral stent was placed, but renal function did not improve. Urinary cytology revealed the presence of decoy cells and polyomavirus was detected in blood and urine by qualitative polymerase chain reaction. Renal biopsy findings were consistent with polyomavirus -associated nephropathy. In the second case, leucopaenia was detected in an asymptomatic patient 6 months after transplantation. Mycophenolate mophetil dosage was reduced but renal allograft function deteriorated, and a kidney biopsy revealed polyomavirus -associated nephropathy, also with SV40 positive cells. In both patients immunosuppression with tacrolimus was reduced, mycophenolate mophetil stopped and intravenous immune globulin plus ciprofloxacin started. As renal function continued to deteriorate, therapy with leflunomide (40 mg/day) was associated and maintained during 5 and 3 months respectively. In the first patient, renal function stabilised within one month of starting leflunomide and polymerase chain reaction was negative for polyomavirus after 5 months. A repeated allograft biopsy 6 months later showed no evidence of polyomavirus nephropathy. In the second patient, polyomavirus was undetectable in blood and urine by polymerase chain reaction after 3 months of leflunomide treatment, with no evidence of polyomavirus infection in a repeated biopsy 6 months after beginning treatment.

A ten-year follow-up of human leptospirosis in Uruguay: an unresolved health problem

Leptospira spp. are delicate bacteria that cannot be studied by usual microbiological methods. They cause leptospirosis, a zoonotic disease transmitted to humans through infected urine of wild or domestic animals. We studied the incidence of this disease in the Uruguayan population, its epidemiologic and clinical features, and compared diagnostic techniques. After examining 6,778 suspect cases, we estimated that about 15 infections/100,000 inhabitants occurred yearly, affecting mainly young male rural workers. Awareness about leptospirosis has grown among health professionals, and its lethality has consequently decreased. Bovine infections were probably the principal source of human disease. Rainfall volumes and floods were major factors of varying incidence. Most patients had fever, asthenia, myalgias or cephalalgia, with at least one additional abnormal clinical feature. 30-40% of confirmed cases presented abdominal signs and symptoms, conjunctival suffusion and altered renal or urinary function. Jaundice was more frequent in patients aged > 40 years. Clinical infections followed an acute pattern and their usual outcome was complete recovery. Laboratory diagnosis was based on indirect micro-agglutination standard technique (MAT). Second serum samples were difficult to obtain, often impairing completion of diagnosis. Immunofluorescence was useful as a screening test and for early detection of probable infections.

Determination of permethrin and its metabolites in hearts and urines of rats by GC-ECD and GC-MS

Pyrethroids are pesticides very used in agriculture, which tend to replace organophosfate and carbamate insecticides. These pesticides have shown to exhibit cardiotoxicity. The aim of this study was to assess if cardiotoxicity is due to direct or indirect effects (metabolites) of permethrin on hearts. There were studies 8 rats; three of them were sacrificed after 24 of the end of treatment with permethrin and the other four were sacrificed after 14 days of the end of treatment. Afterwards hearts and urines were collected. The amounts of permethrin and its main metabolite (3-PBA) were evaluated on hearts and urines of female rats which were treated with permethrin and sacrificed the day after and 14 days after the treatment. Moreover has been highlighted the difference of amount of permethrin and its metabolite between rats sacrificed immediately at the end of treatment and those sacrificed after 14 days. The study of permethrin was accomplished by liquid-liquid extraction and GC-ECD. The evaluation of 3-PBA was performed by SPE procedure with 2-PBA as internal standard and gas-chromatography GC-MS. The concentration of permethrin in hearts is basically the same in 24h and 14 days. The 3-PBA concentration in urines decreased 50 times from 24h to 14 days. In hearts the 3-PBA level also decrease but only 2.24 times and a high variation of results were achieved in rats after 14 days.

Determination of permethrin and its metabolite in brains and urines of rats by GC-ECD and GC-MS

Synthetic pyrethroids are pesticides derived from naturally occurring pyrethrins, taken from pyrethrum of dried Chrysanthemum flowers. Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Many pyrethroids can significantly harm the nervous system. Permethrin could be one of the factors involved in the onset of neurodegenerative diseases. The present study aims to evaluate in brain, the effect that can induce the exposure to permethrin, during early life of female rats (from 6 to 21 days of life). Therefore, have been examined the concentrations of permethrin and its main metabolite (3-PBA) in the brain and urine in female rats sacrificed the day after and 14 days after treating. The different concentrations of permethrin and 3-PBA (after 24h and after 14 days in the end of treatment) were obtained using two different methods. The evaluation of permethrin by liquid-liquid extraction and GC-ECD was performed. The levels of the 3-PBA (in urine and brains) were obtained by SPE procedure and GC-MS using the 2- PBA as internal standard.

MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS

SUMMARYIntroduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals.Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used.Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91.Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

Desenvolvimento de um biossensor para Ácido Glutâmico

A presente dissertação tem com objetivo o desenvolvimento de um biossensor com base nos polímeros de impressão molecular para a deteção de uma molécula alvo, o ácido glutâmico que é convertido em glutamina pela glutamina sintetase, recorrendo à potenciometria. Nas células neoplásicas a glutamina não é sintetizada podendo-se considerar que o ácido glutâmico é um potencial agente anti-cancro. A técnica de impressão molécular utilizada foi a polimerização em bulk, combinando a acrilamida e a bis acrilamida com o ácido glutâmico. Para se verificar se a resposta potenciométrica obtida era de facto da molécula alvo foram preparados em paralelo com os sensores, materiais de controlo, ou seja, moléculas sem impressão molécular (NIP). Para se controlar a constituíção química dos vários sensores nomeadamente, do NIP e do polímero de impressão molecular (MIP) antes e após a remoção bem como a molécula foram realizados estudos de Espetroscopia de Infravermelhos de Transformada de Fourier (FTIR), Scanning electron microscope (SEM) e Espetroscopia de Raios X por dispersão em energia (EDS). Os materiais desenvolvidos foram aplicados em várias membranas que diferiam umas das outras, sendo seletivas ao ião. A avaliação das características gerais das membranas baseou-se na análise das curvas de calibração, conseguidas em meios com pHs diferentes, comparando os vários elétrodos. O pH 5 foi o que apresentou melhor resultado, associado a uma membrana que continha um aditivo, o p-tetra-octilphenol, e com o sensor com percentagem de 3%. Posto isto, testou-se em material biológico, urina, com as melhores características quer em termos de sensibilidade (18,32mV/década) quer em termos de linearidade (1,6x10-6 a 1,48x10-3 mol/L). Verificou-se ainda que aplicando iões interferentes na solução, estes não interferem nesta, podendo ser aplicados na amostra sem que haja alteração na resposta potenciométrica. O elétrodo é capaz de distinguir o ácido glutâmico dos restantes iões presentes na solução.

Magnesium – association with inflammation and renal disease in systemic lupus erythematosus

Introduction: Recent studies suggest that magnesium deficiency may play a role in inflammation. In diabetes and cardio-vascular diseases, conditions with a component of chronic inflammation, C–reactive protein levels are higher and associated with low serum magnesium. The objective of this study is to evaluate serum magnesium levels in patients with systemic lupus erythematosus and its potential association with inflammation and renal manifestations. Methods: All patients with systemic lupus erythematosus followed in a Systemic Immune Diseases Unit, from January 2012 until January 2014, were included in this cross sectional analysis. Patients with infection, neoplasia, liver failure and chronic kidney disease (stage > 3) were excluded. Clinical information and laboratory results (serum magnesium, C-reactive protein, erythrocyte sedimentation rate, serum creatinine and spot urine test) were collected. A multivariate analysis was performed to explore possible predictive factors for hypomagnesaemia. Results: One hundred and two patients were included (94.1% female, 21-86 years). 33.4% had hypertension, 8.8% had diabetes and 20.6% had hypomagnesaemia (< 1.8mg/dL). There were no significant differences between the inflammatory parameters of patients with hypomagnesaemia or normomagnesaemia. Serum magnesium was significantly lower with increasing comorbidities (p = 0.01). Leukocyturia was significantly higher in the hypomagnesaemia group (p = 0.03) and haematuria had a negative correlation with serum magnesium (-0.23, p < 0.05). Multivariate analysis showed that patients with hypertension and diabetes had higher risk of hypomagnesaemia: OR 42.29 (95% CI, 1.43-1249.31). Leukocyturia was also individually and independently associated with hypomagnesaemia: OR 8.37 (95% CI, 1.40-49.97). Conclusion: The presence of hypomagnesaemia in our patients with systemic lupus erythematosus was high. There was no association between the levels of serum magnesium and the inflammatory parameters. Increasing comorbidities and leukocyturia were independent predictors of lower serum magnesium. Finally, the association of leukocyturia and haematuria with lower serum magnesium may suggest a relationship with a higher disease activity.

FREQUENCY, URINALYSIS AND SUSCEPTIBILITY PROFILE OF PATHOGENS CAUSING URINARY TRACT INFECTIONS IN ENUGU STATE, SOUTHEAST NIGERIA

Objective: This study was designed to determine the frequency and causative agent(s) of urinary tract infections (UTIs) in individuals with symptoms of urinary tract infections in Enugu State of Southeast Nigeria, and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture.Methods: The study involved 211 individuals (149 females and 62 males) clinically suspected for UTI. Urine samples were collected by the mid-stream ‘clean catch’ method and tested using standard procedures. Antibiotic susceptibility of the isolated pathogens was tested using the Kirby-Bauer technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.Results: Microscopy of centrifuged urine samples showed 16 patients had pyuria while 54 had pus cells. Calcium oxalate crystals were found in 14 samples. Urinalysis performed with urine samples showed 17 had protein; seven were nitrite positive and three had moderate to high glucose concentration. Fifty-four urine samples (36.2%) from females and 12 (19.4%) from males showed significant growth upon culture. Gram stain and biochemical tests identified nine different organisms with Escherichia coli as the most common isolated species. Forty three randomly selected strains were further tested for their susceptibility against a panel of antibiotics. Thirty isolates (81.08%) were resistant to four or more antibiotics with the highest resistance shown by E. coli (76.67%). All the Gram- negative isolates were resistant to Ampicilox, Cefuroxime and Amoxicillin.Conclusion: Urinary tract infections were found more in females in the area under study. As found in other studies, E. coli was the most predominant isolate, although other organisms seem to be on the increase.

SEROLOGICAL AND MOLECULAR SURVEY OF Leptospira spp. AMONG CART HORSES FROM AN ENDEMIC AREA OF HUMAN LEPTOSPIROSIS IN CURITIBA, SOUTHERN BRAZIL

Introduction: Cart horses are a re-emerging population employed to carry recyclable material in cities. Methods: Sixty-two horses were sampled in an endemic area of human leptospirosis. The microscopic agglutination test (MAT) and real-time polymerase chain reaction (qPCR) were performed. Results: A seropositivity of 75.8% with serovar Icterohaemorrhagiae in 80.8% of the horses was observed. Blood and urine were qPCR negative. MAT showed positive correlations with rainfall (p = 0.02) and flooding (p = 0.03). Conclusions: Although horses may be constantly exposed to Leptospira spp. in the environment mostly because of rainfall and flooding, no leptospiremia or leptospiruria were observed in this study.