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1.
Vet Parasitol ; 274: 108920, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31493694

RESUMO

Visceral leishmaniasis (VL) is a zoonosis caused by the parasite Leishmania infantum and the dog is its main reservoir in rural and urban areas. The diagnosis of infection is mainly based on the presence of anti-Leishmania IgG antibodies in the serum of infected dogs. In this study, the sensitivity and specificity of qualitative rapid tests (RTs) dual path platform (DPP) Bio-Manguinhos, rapid enzyme-linked immunosorbent assay (ELISA) IDEXX, Kalazar Detect and ALERE, as well as quantitative ELISA Bio-Manguinhos and in-house indirect immunofluorescence assay (IFA) tests were analyzed in sera from infected and uninfected dogs. Serial dilutions of the in-house IFA were compared with RTs and ELISA Bio-Manguinhos. The results showed that none of the tests reached 100% sensitivity and specificity. There was no statistical difference between the analyzed RTs. The most sensitive test was the DPP Bio-Manguinhos (97.9%), while the rapid ELISA IDEXX showed higher specificity (100%). In the treatment setting of infected and/or diseased animals, quantitative tests for monitoring the evolution of antibody titers are required, which indicates the maintenance of in-house IFA in animal handling. Furthermore, we demonstrate that the RTs present higher sensitivity in serum samples with superior antibody titers obtained in the in-house IFA. However, the RTs exhibited false negatives in samples with low titers of antibodies. Among the RTs, only the DPP Bio-Manguinhos presented better performance in this situation. Therefore, the use of RTs for the diagnosis of VL in dogs with low titers of antibodies, such as asymptomatic, should be carefully evaluated.


Assuntos
Doenças do Cão/sangue , Leishmania infantum , Leishmaniose Visceral/veterinária , Testes Sorológicos/veterinária , Animais , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Sensibilidade e Especificidade
2.
Exp Parasitol ; 203: 23-29, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150654

RESUMO

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.


Assuntos
Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Pele/parasitologia , Animais , Sequência de Bases , Colorimetria , Cricetinae , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Masculino , Mesocricetus , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Coloração pela Prata
3.
Future Microbiol ; 13: 429-436, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29125786

RESUMO

AIM: To produce and test recombinant multiepitope proteins as an alternative assay for the serological diagnosis of cryptococcosis. MATERIALS & METHODS: Previously, synthetic peptides were used to detect anti-Cryptococcus antibodies, and in silico analyses showed that the union of peptides would improve the results. Here, the coding sequences of these peptides were assembled into synthetic genes. Four genes have been cloned and expressed in Escherichia coli, producing recombinant multiepitope proteins: proteins A, B, C and D. RESULTS: All constructs yielded good results; however, protein D showed the best results, with a sensitivity of 88.57% and specificity of 100%. CONCLUSION: The multiepitope proteins were shown to be potential antigens for the diagnosis of cryptococcosis in an attempt to detect anti-Cryptococcus antibodies.


Assuntos
Anticorpos Antifúngicos/imunologia , Criptococose/diagnóstico , Cryptococcus/imunologia , Epitopos de Linfócito B/imunologia , Proteínas Recombinantes/imunologia , Sequência de Aminoácidos , Anticorpos Antifúngicos/sangue , Antígenos de Fungos/genética , Antígenos de Fungos/imunologia , Criptococose/sangue , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus/genética , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/genética , Escherichia coli/genética , Humanos , Proteínas Recombinantes/genética , Sensibilidade e Especificidade
4.
Autoimmunity ; 50(4): 247-256, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28675715

RESUMO

Systemic lupus erythematosus (SLE) is an autoimmune disease of the connective tissue with a large spectrum of clinical manifestations. Immune deregulation leads to autoantibody and immune complexes overproduction, complement activation, and persistent tissue inflammation. Considering that the current diagnosis depends on the interpretation of the complex criteria established by the American College of Rheumatology and that the disease course is characterized by unpredictable activations and remissions, each patient develops different manifestations, and therefore, the discovery of specific biomarkers is urgently required. Therefore, this study aimed to identify putative biomarkers for active and inactive SLE potentially capable in distinguishing laboratorial SLE from other autoimmune diseases. The 2D-DIGE proteomics technique was used to evaluate the differential abundance of proteins between patients with active SLE, inactive SLE, patients with other autoimmune disease, and healthy individuals. Six proteins showed increased abundance in active SLE (A) and inactive SLE (I) compared to the C and O groups, but not between groups A and I. There were two transthyretin (TTR) fragments or proteins with a structure similar to TTR (accession numbers: PDB: 1GKO_A and 2PAB_A), retinol-binding protein 4 (RBP4) isoform X1 (no information in databases such as UNIPROT), and antibody fragments. Two proteins, APO-AIV and SP-40,40, were upregulated in group A than in O and C and in group I versus C, but not in group I versus O. Therefore, we suggest these proteins to be considered as candidates for the diagnosis of SLE.


Assuntos
Biomarcadores , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , Espectrometria de Massas , Eletroforese em Gel Diferencial Bidimensional , Adulto , Doenças Autoimunes/sangue , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem
5.
Vaccine ; 34(19): 2233-9, 2016 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-26997002

RESUMO

BACKGROUND: A canine vaccine remains a promising approach for effective control of visceral leishmaniasis (VL), given its complex epidemiology in areas where zoonotic VL is prevalent. Leish-Tec(®) is a recombinant vaccine, based on the Leishmania A2 antigen, against canine VL (CVL). It is, since 2014, the single commercial vaccine licensed in Brazil. Here, Leish-Tec(®) efficacy was estimated through a randomized field trial (RFT), in a highly VL endemic area. METHODS: The RFT was conducted from 2008 to 2010 in an endemic area of southeastern Brazil, presenting a CVL seroprevalence of 41.9%. Eight hundred forty-seven seronegative dogs were randomly selected to receive Leish-Tec(®) (n=429) or placebo (n=418). Animals were followed up by clinical, serological, and parasitological exams for 18 months. The CVL incidence in both groups was compared through proportion analysis. RESULTS: A significant reduction in the number of cases of CVL was observed in the vaccine group, as compared with the placebo group, whether efficacy was estimated according to parasitological results (71.4%; 95% CI: 34.9-87.3%; p=0.001; risk ratio=0.287), by adding results of xenodiagnosis and parasitological exams (58.1%; 95% CI: 26.0-76.3%; p=0.002; risk ratio=0.419). Among the animals that converted to a positive anti-A2 serology, efficacy reached 80.8% (95% CI: 37.6-94.1%, p=0.001; risk ratio=0.192). Xenodiagnosis has detected a reduction of 46.6% (p=0.05) in transmission to sand flies from vaccinated animals presenting anti-A2 positive serology. CONCLUSION: The Leish-Tec(®) vaccine proved significantly effective for prophylaxis of CVL, after natural challenge assured by transmission of Leishmania parasites, in a highly endemic area. Noteworthy, this report has unveiled the complexity of performing a RFT for anti-CVL vaccines in Brazil, which may be helpful for designing of future studies.


Assuntos
Antígenos de Protozoários/imunologia , Doenças do Cão/prevenção & controle , Vacinas contra Leishmaniose/uso terapêutico , Leishmaniose Visceral/veterinária , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Brasil , Doenças do Cão/parasitologia , Cães , Imunidade Humoral , Leishmaniose Visceral/prevenção & controle , Psychodidae/parasitologia , Xenodiagnóstico
6.
Future Microbiol ; 9(7): 871-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25156376

RESUMO

AIM: To determine the immunoreactivity of synthetic Cryptococcus-derived peptides. MATERIALS & METHODS: A total of 63 B-cell epitopes from previously identified Cryptococcus gattii immunoreactive proteins were synthesized and evaluated as antigens in ELISAs. The peptides were first evaluated for their ability to react against sera from immunocompetent subjects carrying cryptococcal meningitis. Peptides that yielded high sensitivity and specificity in the first test were then retested with sera from individuals with other fungal pathologies for cross-reactivity determination. RESULTS: Six of 63 synthetic peptides were recognized by antibodies in immunoassays, with a specificity of 100%, sensitivity of 78% and low cross-reactivity. CONCLUSION: We successfully determined the immunoreactivity of selected synthetic peptides of C. gattii derived proteins.


Assuntos
Proteínas de Bactérias/imunologia , Cryptococcus gattii/imunologia , Peptídeos/imunologia , Anticorpos Antifúngicos/análise , Anticorpos Antifúngicos/imunologia , Proteínas de Bactérias/genética , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus gattii/genética , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/imunologia , Humanos , Peptídeos/síntese química , Peptídeos/química
7.
PLoS One ; 9(5): e97526, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24842666

RESUMO

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.


Assuntos
Proteínas de Protozoários/análise , Radiação Ionizante , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/efeitos da radiação , Eletroforese em Gel Bidimensional , Proteômica
8.
Acta Trop ; 137: 25-30, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24801885

RESUMO

Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens' sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Portador Sadio/veterinária , Doenças do Cão/diagnóstico , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Animais , Antígenos de Protozoários/genética , Brasil , Portador Sadio/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Leishmania infantum/genética , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Testes Sorológicos/métodos
9.
J Proteome Res ; 13(4): 1860-72, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24617796

RESUMO

Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite-host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron-sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase.


Assuntos
Leishmania infantum/metabolismo , Leishmania infantum/patogenicidade , Proteínas de Protozoários/análise , Fatores de Virulência/análise , Animais , Células Cultivadas , Cricetinae , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Proteoma/análise , Proteômica/métodos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo
10.
Anaerobe ; 22: 69-76, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23618673

RESUMO

Bacteroides fragilis is the anaerobe most frequently isolated from clinical specimens and piperacillin/tazobactam is among the drugs that can be used to treat polymicrobial infections in which this bacteria is often involved. During antibiotic therapy, inhibitory concentrations of antibiotics are always followed by subinhibitory concentrations which can generate phenotypic changes in bacteria. So, in this study we aimed to evaluate changes in the proteomic profile of B. fragilis grown in a sub-MIC of PTZ, using 2-D electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight/time of-flight. Analysis of the 2-DE gels showed 18 spots with significantly different volume percentages between experimental conditions and 12 were successfully identified by MS/MS. Two proteins with decreased abundance in sub-MIC condition were involved in the glycolysis (glyceraldehyde-3-phosphate dehydrogenase and triose phosphate isomerase), others two involved in amino acid metabolism (Oxoacyl-(acyl-carrier protein) synthase II and dihydrodipicolinate reductase), and finally, one protein involved in fatty acid metabolism (UDP-N-acetylglucosamine acyltransferase). Among the proteins with increased abundance, we founded three ATP synthase (alpha, beta, and alpha type V), which could be involved in antibiotic bacterial resistance by efflux pump, one protein involved in glycolysis (enolase), and one involved in protein degradation (aminoacyl-histidine dipeptidase). In conclusion, our data show overall changes in the proteome of B. fragilis conducted by sub-MIC of PTZ, whose consequences on bacterial physiology deserve further investigation.


Assuntos
Antibacterianos/farmacologia , Bacteroides fragilis/química , Bacteroides fragilis/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/farmacologia , Piperacilina/farmacologia , Combinação Piperacilina e Tazobactam , Proteômica
11.
Future Microbiol ; 8(4): 549-63, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23534365

RESUMO

AIM: To identify immunoreactive proteins of Cryptococcus gattii genotype VGII and their B-cell epitopes. MATERIALS & METHODS: We combined 2D gel electrophoresis, immunoblotting and mass spectrometry to identify immunoreactive proteins from four strains of C. gattii genotype VGII (CG01, CG02, CG03 and R265). Next, we screened the identified proteins to map B-cell epitopes. RESULTS: Sixty-eight immunoreactive proteins were identified. The strains and the number of proteins we found were: CG01 (12), CG02 (12), CG03 (18) and R265 (26). In addition, we mapped 374 peptides potentially targeted by B cells. CONCLUSION: Both immunoreactive proteins and B-cell epitopes of C. gattii genotype VGII that were potentially targeted by a host humoral response were identified. Considering the evolutionary relevance of the identified proteins, we may speculate that they could be used as the initial targets for recombinant protein and peptide synthesis aimed at the development of immunodiagnostic tools for cryptococcosis.


Assuntos
Antígenos de Fungos/análise , Criptococose/diagnóstico , Criptococose/microbiologia , Cryptococcus gattii/química , Cryptococcus gattii/imunologia , Proteínas Fúngicas/química , Proteômica , Antígenos de Fungos/genética , Antígenos de Fungos/imunologia , Cryptococcus gattii/genética , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Humanos , Espectrometria de Massas
12.
Trans R Soc Trop Med Hyg ; 107(4): 212-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23423433

RESUMO

BACKGROUND: The eco-epidemiological complexity of American cutaneous leishmaniasis (ACL) has made it difficult to devise an efficient strategy for management of the disease, and development of an effective vaccine remains the most promising approach. The objective of the study was to determine the reduction in incidence of ACL following intramuscular administration of two doses of a killed Leishmania (Leishmania) amazonensis vaccine. METHODS: A cluster randomised trial was conducted from 2002 to 2011 in 108 localities in an endemic area of southeast Brazil. Communities were stratified according to population size, and randomly allocated to receive vaccine (n = 50) or placebo (n = 58). The post-vaccination ACL incidence rates in the two groups were compared through covariance analysis. RESULTS: A cyclic fluctuation in the number of cases recorded during the 18-year pre-vaccination period was similar in both groups. Following the vaccination campaign, a significant reduction in the number of cases of ACL was observed in the vaccine group compared with the placebo group. This group also included the individuals who refused to participate in the trial. CONCLUSION: This study demonstrated that the vaccine has been able to confer protection against ACL up to the present time. It is necessary to continue epidemiological surveillance to determine the duration of the vaccine's effectiveness.


Assuntos
Vacinas contra Leishmaniose , Leishmaniose Cutânea/prevenção & controle , Vacinação em Massa/métodos , Adolescente , Adulto , Brasil/epidemiologia , Criança , Análise por Conglomerados , Feminino , Humanos , Incidência , Injeções Intramusculares , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Cutânea/epidemiologia , Masculino , Adulto Jovem
13.
Res Microbiol ; 161(4): 268-75, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20381611

RESUMO

The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. In addition to the difficulties in controlling infectious diseases, the phenotype of resistance can generate metabolic changes which, in turn, can interfere with host-pathogen interactions. The aim of the present study was to identify changes in the subproteome of a laboratory-derived piperacillin/tazobactam-resistant strain of Escherichia coli (minimal inhibitory concentration [MIC] = 128 mg/L) as compared with its susceptible wild-type strain E. coli ATCC 25922 (MIC = 2 mg/L) using 2-D fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF MS). In the resistant strain, a total of 12 protein species were increased in abundance relative to the wild-type strain, including those related to bacterial virulence, antibiotic resistance and DNA protection during stress. Fourteen proteins were increased in abundance in the wild-type strain compared to the resistant strain, including those involved in glycolysis, protein biosynthesis, pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. In conclusion, our data show overall changes in the subproteome of the piperacillin/tazobactam-resistant strain, reporting for the first time the potential role of a multidrug efflux pump system in E. coli resistance to piperacillin/tazobactam.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Ácido Penicilânico/análogos & derivados , Piperacilina/farmacologia , Proteômica , Eletroforese em Gel Bidimensional , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Ácido Penicilânico/farmacologia , Tazobactam
14.
Rev Soc Bras Med Trop ; 39(4): 385-7, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17119757

RESUMO

While testing 414 sera for the diagnosis of Chagas' disease, the conventional reactions of indirect hemagglutination, indirect immunofluorescence and the immunosorbent assay showed a sensitivity of 95.7%, 100% and 98.2% and a specificity of 98%, 98% and 96.4%, respectively, and an excellent association using Fisher's exact test. Chemiluminescence presented 100% sensitivity and 89.6% specificity, while PCR showed 100% specificity and 1.2% sensitivity. It is believed that the three conventional serological reactions are still adequate for diagnosing Chagas' disease.


Assuntos
Doença de Chagas/diagnóstico , Reação em Cadeia da Polimerase , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Testes de Hemaglutinação , Humanos , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia
15.
BMC Vet Res ; 2: 17, 2006 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-16762067

RESUMO

BACKGROUND: We compared skin biopsy samples from different anatomical regions for detecting Leishmania in dogs, using histological (HE), immunohistochemical (IHC) and polymerase chain reaction (PCR) techniques. RESULTS: The sensitivity was 82.8 percent for PCR, 62.1 percent for IHC and 44.8 percent for HE. These methods do not appear to depend on the clinical status of the animal or the anatomical source of the skin sample; there is no "best region" for any method. However, PCR was more effective than IHC and HE for ear and nose skin samples whereas IHC was better than HE for nose samples. There was weak agreement between PCR and HE for all tissue samples; good agreement between PCR and IHC for ear and abdomen samples, and weak agreement for nose; and optimal agreement between IHC and HE for ear and abdomen and good agreement for nose samples. CONCLUSION: The PCR on ear skin could be the best procedure for diagnosing canine visceral leishmaniasis. The good agreement between PCR and IHC indicates that IHC can be used as an alternative method. Finally, tissue samples from ears, nose and abdomen, particularly ears and nose, are potentially useful for diagnosing canine visceral leishmaniasis independently of the animal's clinical status.


Assuntos
Biópsia/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Imuno-Histoquímica/veterinária , Leishmaniose Cutânea/veterinária , Reação em Cadeia da Polimerase/veterinária , Pele/parasitologia , Animais , Antígenos de Protozoários/análise , Cães , Imuno-Histoquímica/métodos , Leishmaniose Cutânea/diagnóstico , Inclusão em Parafina , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
16.
Vet Parasitol ; 140(3-4): 231-8, 2006 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-16682124

RESUMO

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania (Leishmania) chagasi and Leishmania (Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (> or = 1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic (n = 12) and asymptomatic (n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. (L.) chagasi except one, infected with L. (V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR-RFLP can be used to identify Leishmania species in dog tissue imprint stained slides.


Assuntos
Doenças do Cão/diagnóstico , Leishmania braziliensis/isolamento & purificação , Leishmania/isolamento & purificação , Leishmaniose Cutânea/veterinária , Leishmaniose Visceral/veterinária , Polimorfismo de Fragmento de Restrição , Animais , Sequência de Bases , Brasil , DNA de Protozoário/química , Diagnóstico Diferencial , Cães , Feminino , Técnica Indireta de Fluorescência para Anticorpo/métodos , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Leishmania/genética , Leishmania donovani/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Visceral/diagnóstico , Masculino , Especificidade de Órgãos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade
17.
Acta Trop ; 98(1): 87-93, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16527224

RESUMO

Montenegro skin test (MST) represents the main complementary diagnostic test for tegumentary leishmaniases (TL) in endemic regions. Most antigen formulations used for the MST contain thimerosal as preservative. The Food and Drug Administration (FDA), however, recommended reducing or eliminating thimerosal from vaccines and other biological reagents and the Agência Nacional de Vigilância Sanitária (ANVISA) in Brazil, prohibited the use of mercurial compounds in immunobiologicals. In the search for an alternative stabilizer, phenol and thimerosal were tested as antigen preservatives in MST. Formulations were tested when fresh and after a 12-month storage at 4 degrees C in TL confirmed mice and human patients, and were evaluated for protein constitution by SDS-PAGE, Western blot and anti-gp63 ELISA. In mice, a decrease in the diagnostic effectiveness in merthiolate formulation was observed after a 12-month storage. SDS-PAGE, Western blot and anti-gp63 ELISA analyses showed a degradation of antigen proteins in both formulations after 12-month storage and that phenol-preserved antigen was quantitatively and qualitatively better than the merthiolate-preserved one. In patients, the average of induration diameter was larger in fresh antigens (p<0.05). However, storage time did not jeopardize their diagnostic capacity. No non-specific reactions produced by phenol or merthiolate were observed neither in humans nor in mice. Phenol could be a good alternative to replace the merthiolate in MST, and despite the proteolytic activity, antigens remain viable for at least 12 months.


Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Leishmaniose Cutânea/diagnóstico , Fenol/química , Conservantes Farmacêuticos/química , Testes Cutâneos/métodos , Timerosal/química , Animais , Armazenamento de Medicamentos , Feminino , Humanos , Leishmaniose Cutânea/imunologia , Camundongos , Fenol/farmacologia , Conservantes Farmacêuticos/farmacologia , Timerosal/farmacologia
18.
Rev Soc Bras Med Trop ; 37(1): 15-7, 2004.
Artigo em Português | MEDLINE | ID: mdl-15042175

RESUMO

Vaccination using surface antigen from hepatitis B virus has not been successfully responded by hemodialysis patients. The present study was aimed at assessing a possible relationship between human leukocyte antigens and the low production of protective antibodies (anti-HbS) against the surface antigen from hepatitis B by patients with chronic renal failure submitted to hemodialysis programs. The antigens HLA-DR and HLA-DQ were identified in 76 hemodialysis patients through classic microlymphotoxicity. Our results showed that 34.2% of the patients were non-responsive to the vaccine VHB. The most frequent HLA specificity were: HLA-DR3, DR-7 and DQ2 with a significant association for HLA-DR3 (p=0.0025; OR 5.1; IC 95% 1.36-19.10). Such data suggest an association between genes from HLA class II antigens and the humoral non-response to the vaccine VHB.


Assuntos
Antígenos HLA/imunologia , Anticorpos Anti-Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Hepatite B/prevenção & controle , Epitopos , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/imunologia , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/administração & dosagem , Humanos , Falência Renal Crônica/terapia , Fenótipo , Diálise Renal/efeitos adversos
19.
Mem Inst Oswaldo Cruz ; 98(8): 1021-3, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15049083

RESUMO

The present paper reports a laboratory investigation performed between the years of 2000 and 2002 to study a virological surveillance program introduced in the state of Piauí to support an epidemiological survey of the disease. Dengue virus type 3 (DENV-3) existence in the state was detected in May 2002 when a high number of dengue cases due to DENV-1 and DENV-2 were reported. An assessment on the population knowledge about the disease and its transmission showed that almost 50% of the population were still unaware of the epidemiological features of dengue.


Assuntos
Vírus da Dengue/classificação , Dengue/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil/epidemiologia , Criança , Pré-Escolar , Dengue/virologia , Vírus da Dengue/genética , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos e Questionários
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