Detecção de sífilis por ensaios de ELISA e VDRL em doadores de sangue do Hemonúcleo de Guarapuava, Estado do Paraná

O objetivo desse estudo foi avaliar a soroprevalência de sífilis em 5.752 doadores de sangue atendidos no Hemonúcleo de Guarapuava-PR, em 2006. As taxas de positividade foram de 2,1% pelo teste de ensaio imunoenzimático e 0,2% pelo Veneral Disease Research Laboratory, mostrando baixa prevalência de sífilis nos indivíduos que procuraram este banco de sangue.

Comportamento dos testes sorológicos ML Flow e ELISA (PGL-I) em áreas endêmica e não endêmica de hanseníase

O ML Flow e o ELISA PGL-I são testes sorológicos que detectam anticorpos IgM contra o glicolipídio fenólico I específico do Mycobacterium leprae. Para avaliar o comportamento destes testes em áreas endêmica e não endêmica para hanseníase foram estudados 351 voluntários no Brasil e no Chile, incluindo pacientes com hanseníase, controles sadios, portadores de outras doenças infecciosas, não infecciosas e dermatoses que fazem diagnóstico diferencial com hanseníase. O ponto de corte do ELISA foi estabelecido pelo método da Curva ROC (> 0,157). Em área endêmica, o ML Flow apresentou resultados positivos em 70% dos pacientes com hanseníase; o ELISA foi positivo em 53,3%. Em área não endêmica, o ML Flow foi negativo em todos os voluntários testados; o ELISA foi positivo em 4 voluntários. O ML Flow é um ensaio mais rápido, facilmente aplicável e, portanto, mais adequado para ser utilizado na Atenção Básica; o ELISA necessita, alem de uma infra-estrutura de laboratório adequada, pessoal treinado e especializado em sua execução.

Pesquisa de anticorpos anti PGL-I através de ELISA em tatus selvagens do Brasil

Tatus têm sido envolvidos na transmissão da hanseníase e considerados como fonte de Mycobacterium leprae em muitas publicações. Médicos de partes dos EUA consideram o contato com tatus um fator de risco para hanseníase. Entretanto, há um desafio associado ao papel do tatu na perpetuação da hanseníase no Continente Americano. Foi pesquisada a presença de anticorpos anti-PGL-I em tatus selvagens de áreas endêmicas em hanseníase do Estado do Espírito Santo, Brasil, através de ELISA realizado em amostras de soro de 47 animais. Elisa positivo foi encontrado em 5 (10.6%) tatus. Tatus infectados podem ter algum papel na transmissão da hanseníase disseminando bacilos no meio ambiente, talvez tornando mais difícil a interrupção da cadeia de transmissão e redução do número de casos novos de hanseníase. A técnica de ELISA é um eficiente método para investigação soroepidemiológica da presença do Mycobacterium leprae em tatus.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

Increased detection of schistosomiasis with Kato-Katz and SWAP-IgG-ELISA in a Northeastern Brazil low-intensity transmission area

INTRODUCTION: The laboratory diagnosis of schistosomiasis is based mainly on the detection of parasite eggs in stool samples through the Kato-Katz (KK) technique, reading one slide by test. However, a widely known limitation of parasitological methods is reduced sensitivity, particularly in low endemic areas. METHODS: To increase sensitivity, we conducted further slide readings from the same stool sample using the parasitological method associated with a serological test. We used the KK method (three slides) and the IgG anti-Schistosoma mansoni-enzyme-linked immunosorbent assay (ELISA) technique to diagnose schistosomiasis in low endemic areas in the Brazilian State of Ceará. Fecal samples and sera from 250 individuals were analyzed. RESULTS: Sixteen percent and 47.2% of samples were positive in parasitological tests and serological tests, respectively. Parasitological methods showed that 32 (80%) individuals tested positive on the first slide, 6 (15%) on the second slide, and 2 (5%) on the third. The performance of the ELISA test in the diagnosis, using the KK method as diagnostic reference, showed a negative predictive value of 100%, with specificity and positive predictive values of 62.8% and 33.9%, respectively. CONCLUSIONS: In this study, the increase from one to three slides analyzed per sample using the KK technique was shown to be a useful procedure for increasing the diagnostic sensitivity of this technique.

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS : This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.

Assessment of a protease inhibitor peptide for anti-ageing

Ageing and skin exposure to UV radiation induces production and activation of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE). These enzymes are known to break down the extracellular matrix (ECM) which leads to wrinkle formation. Here, we demonstrated the potential of a solid-in-oil nanodispersion containing a competitive inhibitor peptide of HNE mixed with hyaluronic acid (HA), displaying 158 nm of mean diameter, to protect the skin against the ageing effects. Western blot analysis demonstrated that activation of MMP-1 in fibroblasts by HNE treatment is inhibited by the solid-in-oil nanodispersion containing the peptide and HA. The results clearly demonstrate that solid-in-oil nanodispersion containing the HNE inhibitor peptide is a promising strategy for anti-ageing effects. This effect can be seen particularly by ECM regulation by affecting fibroblasts. The formulation also enhances the formation of thicker bundles of actin filaments.

The effects of solvent composition on the affinity of a peptide towards hair keratin: experimental and molecular dynamics data

The study of the interaction between hair filaments and formulations or peptides is of utmost importance in fields like cosmetic research. Keratin intermediate filaments structure is not fully described, limiting the molecular dynamics (MD) studies in this field although its high potential to improve the area. We developed a computational model of a truncated protofibril, simulated its behavior in alcoholic based formulations and with one peptide. The simulations showed a strong interaction between the benzyl alcohol molecules of the formulations and the model, leading to the disorganization of the keratin chains, which regress with the removal of the alcohol molecules. This behavior can explain the increase of peptide uptake in hair shafts evidenced in fluorescence microscopy pictures. The model developed is valid to computationally reproduce the interaction between hair and alcoholic formulations and provide a robust base for new MD studies about hair properties. It is shown that the MD simulations can improve hair cosmetic research, improving the uptake of a compound of interest.

Improved burn wound healing using a bioactive peptide

[Excerpt] Antimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. We took advantage of a commercially available hydrogel, Carbopol, a vehicle for topical administration that maintains a moist environment within the wound site. We hypothesized that the incorporation of LLKKK18 conjugated to dextrin would improve the healing process in rat burns. Whereas the hydrogel improves healing, LLKKK18 released from the dextrin conjugates further accelerates wound closure, and simultaneously improving the quality of healing. Indeed, the release of LLKKK18 reduces oxidative stress and inflammation (low neutrophil and macrophage infiltration and pro-inflammatory cytokines levels). Importantly, it induced a faster resolution of the inflammatory stage through early M2 macrophage recruitment. In addition, LLKKK18 stimulates angiogenesis (increased VEGF and microvessel development in vivo), potentially contributing to more effective transport of nutrients and cytokines. Moreover, collagen staining evaluated by Masson’s Trichrome was visually much more intense after treatment with LLKKK18, suggesting higher collagen deposition. (...)

Synthesis of new peptide derivatives that self-assemble into nanostructured hydrogels for biomedical applications

Tese de Doutoramento em Ciências (área de especialização em Química)

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

The Role of B-Type Natriuretic Peptide in the Diagnosis of Congestive Heart Failure in Patients Presenting to an Emergency Department with Dyspnea

OBJECTIVE: To determine the utility of B-type natriuretic peptide (BNP) in the diagnosis of congestive heart failure (CHF) in patients presenting with dyspnea to an emergency department (ED). METHODS: Seventy patients presenting with dyspnea to an ED from April to July 2001 were included in the study. Mean age was 72±16 years and 33 (47%) were male. BNP was measured in all patients at the moment of admission to the ED. Emergency-care physicians, blinded to BNP values, were required to assign a probable initial diagnosis. A cardiologist retrospectively reviewed the data (blinded to BNP measurements) and assigned a definite diagnosis, which was considered the gold standard for assessing the diagnostic performance of BNP. RESULTS: The mean BNP concentration was higher in patients with CHF (n=36) than in those with other diagnoses (990±550 vs 80±67 pg/mL, p<0.0001). Patients with systolic dysfunction had higher BNP levels than those with preserved systolic function (1,180±641 vs 753±437 pg/mL, p=0.03). At a blood concentration of 200 pg/mL, BNP showed a sensitivity of 100%, specificity of 97.1%, positive predictive value of 97.3%, and negative predictive value of 100%. The application of BNP could have potentially corrected all 16 cases in which the diagnosis was missed by the emergency department physician. CONCLUSION: BNP measurement is a useful tool in the diagnosis of CHF in patients presenting to the ED with dyspnea.

Antígeno heterólogo no diagnóstico imunológico da wuchereriose: extrato de Setaria equina utilizado na reação imunoenzimática - ELISA

A reação imunoenzimática - ELISA foi empregada na sorologia da Wuchereriose, utilizando como antígeno extrato bruto de verme adulto de Setaria equina. Foram estudados soros de 139 indivíduos em três grupos: grupo 1 - de pacientes com diagnóstico parasitológico de Wuchereriose; grupo 2 - de pacientes com diversas patologias; grupo 3 - de pessoas clinicamente normais, de área endêmica e não endêmica. O antígeno utilizado mostrou alta comunidade antigênica coma W. bancrofti. As reações cruzadas, obtidas em particular com soros de pacientes com parasitoses intestinais (áscaris, ancilóstoma) recomendam a investigação de frações antigênicas de Setaria equina que possam fornecer testes de maior especificidade.