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1.
Recife; s.n; 2015. 86 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-983377

ABSTRACT

O exame parasitológico por Kato-Katz ainda é considerado o padrão ouro no diagnóstico da esquistossomose mansônica, entretanto, este apresenta baixa sensibilidade para utilização em inquéritos epidemiológicos. Além disso, as técnicas de diagnóstico imunológico, apresentam reações cruzadas com outros helmintos, protozoários e até bactérias como ocorre com a utilização dos extratos brutos do parasita. Nesse sentido, salientamos que Abath et al. identificaram um peptídeo de 15kDa denominado Sm15, que apresentou uma boa reatividade com soros de animais infectados pelo verme e, portanto, possui potencial para abordagens imunoprofiláticas e para testes diagnósticos. Neste estudo obtivemos o polipeptídio recombinante Sm15 em Escherichia coli e verificamos seu potencial para realização do diagnóstico a partir de amostras de soros de pacientes com diferentes manifestações clínicas da esquistossomose. Através de ELISA constatamos que o Sm15 apresentou maior reatividade frente a soros de pacientes esquistossomóticos, quando comparado ao extrato bruto SEA (P=0.0043). O Sm15 ainda demonstrou melhor desempenho ao apresentar maiores valores de sensibilidade, especificidade e área abaixo da curva ROC (P=0.0030). Além disso, o Sm15 foi capaz de diferenciar pacientes esquistossomóticos quanto à forma clínica, aguda ou crônica (P=0.0007). Os resultados obtidos neste estudo indicam, além de ratificar o potencial diagnóstico apresentado pelo polipeptídeo Sm15, que o mesmo poderá ser capaz de gerar uma alternativa de imunodiagnóstico de elevada acurácia, suprindo assim as lacunas existentes com relação aos testes parasitológicos e sorológicos atualmente disponíveis. Além disso, possibilitará o diagnostico precoce da esquistossomose, realização de inquéritos epidemiológicos em áreas de baixa endemicidade, impedindo assim a evolução da doença para formas clínicas de maior gravidade.


The parasitological examination by Kato-Katz still considered the gold standard in the diagnosis of schistosomiasis, however, it has low sensitivity for use in epidemiological surveys. Moreover, the techniques of immunological diagnosis, have cross-reactivity with other helminth, protozoa and even bacteria as occur with the use of crude parasite extracts. In this regard, we note that Abath and colleagues identified a 15kDa peptide termed SM15, which showed good reactivity with sera from animals infected by the worm, and therefore has potential immunoprophylactic and diagnostic testing approaches. In this study we obtained the recombinant polypeptide in Escherichia coli SM15 and check its potential for making the diagnosis from samples of patient sera with different clinical manifestations of schistosomiasis. By ELISA we found that the SM15 showed higher reactivity towards sera from schistosomiasis patients, when compared to the crude extract SEA (P = 0.0043). The SM15 also demonstrated better performance by presenting higher sensitivity, specificity, and area under the ROC curve (P = 0.0030). In addition, the SM15 was able to differentiate schistosomiasis patients about the clinical presentation, acute or chronic (P = 0.0007). The results of this study indicate not only ratifies the diagnostic potential presented by the SM15 polypeptide, that it may be able to generate an immunodiagnostic alternative high accuracy, thereby supplying the gaps in the parasitological and serological tests currently available. Also, it enables the early diagnosis of schistosomiasis, carrying out epidemiological surveys in low endemicity areas, thereby preventing disease progression to more severe clinical forms.


Subject(s)
Humans , Animals , Immunologic Tests/classification , Immunologic Tests/methods , Recombinant Proteins , Recombinant Proteins/isolation & purification , Schistosomiasis , Antigens, Helminth/genetics , Enzyme-Linked Immunosorbent Assay/methods , Helminth Proteins/genetics , Helminth Proteins/immunology , Schistosoma mansoni/genetics , Sensitivity and Specificity
2.
Article in English | WPRIM | ID: wpr-155349

ABSTRACT

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient's serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.


Subject(s)
Adult , Animals , Antibodies, Helminth/blood , Antigens, Helminth/genetics , Base Sequence , Echinococcosis, Hepatic/immunology , Echinococcosis, Pulmonary/diagnosis , Echinococcus granulosus/genetics , Echinococcus multilocularis/genetics , Electron Transport Complex IV/genetics , Female , Humans , Mitochondria/genetics , Molecular Sequence Data , Republic of Korea , Sequence Analysis, DNA
3.
Rev. Inst. Med. Trop. Säo Paulo ; 49(3): 187-189, May-June 2007. tab, graf
Article in English | LILACS | ID: lil-454767

ABSTRACT

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.


Considerando a escassez de antígenos quimicamente definidos, realmente úteis e confiáveis para aplicação na soroepidemiologia da esquistossomose em larga escala, foi proposto, neste trabalho, um método alternativo para a seleção de clones de cDNA que expressam proteínas com putativo potencial diagnóstico na esquistossomose. Empregando anticorpos específicos contra uma fração proteica de 31/32 kDa (Sm31/32), purificados através da dissociação de imunocomplexos, foram selecionados cinco clones de cDNA a partir de genoteca de verme adulto de Schistosoma mansoni. O seqüenciamento parcial destes clones demonstrou que todos eram relacionados ao S. mansoni: dois apresentaram homologia com a proteína de choque térmico de 70 kDa e os demais com glutationa S-transferase, "homeodomain protein" e uma etiqueta de seqüência expressa (EST). Este último foi o clone que melhor reagiu, durante o processo de seleção, com os anticorpos anti-Sm31/32 dissociados de imunocomplexos. Baseado na seqüência de aminoácidos deste clone, dois peptídeos foram quimicamente sintetizados e analisados separadamente frente a misturas de soros de indivíduos normais e de pacientes com esquistossomose mansoni. Ambos os peptídeos demonstraram uma intensa reatividade somente contra a mistura de soros positivos, sugerindo que estes peptídeos podem ser úteis como antígenos para o diagnóstico da esquistossomose mansoni.


Subject(s)
Humans , Animals , DNA, Complementary/genetics , Peptide Library , Schistosoma mansoni/genetics , Schistosoma mansoni/immunology , Schistosomiasis mansoni/diagnosis , Antibodies, Helminth/genetics , Antibodies, Helminth/immunology , Antigens, Helminth/genetics , Antigens, Helminth/immunology , Cloning, Molecular/methods , DNA, Complementary/immunology , Expressed Sequence Tags , Gene Library , /immunology , Open Reading Frames
4.
Article in English | WPRIM | ID: wpr-59380

ABSTRACT

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.


Subject(s)
Sequence Alignment , Recombinant Proteins/biosynthesis , Phylogeny , Paragonimus westermani/enzymology , Molecular Sequence Data , Hemoglobins/metabolism , Escherichia coli/enzymology , DNA, Complementary/genetics , Cysteine Endopeptidases/genetics , Astacoidea/parasitology , Antigens, Helminth/genetics , Animals , Amino Acid Sequence
5.
Southeast Asian J Trop Med Public Health ; 2003 ; 34 Suppl 2(): 98-102
Article in English | IMSEAR | ID: sea-32150

ABSTRACT

Serodiagnosis by immunoblot, using recombinant chimeric T. solium antigen and native glycoprotein antigens, has been applied for neurocysticercosis cases. Specific antibodies against both antigens were detected in serum samples from NCC patients involving multiple cysts in the brain, whereas it was not always easy to detect specific antibodies in NCC cases with a solitary cyst or calcified lesion(s). On the other hand, the diagnosis for human taeniasis or worm carriers has been routinely performed by stool examination. In this study, multiplex PCR has been established to differentiate taeniasis using Taenia mitochondrial DNA in fecal samples from worm carriers. Furthermore, the molecular identification of human taeniid cestodes by base excision sequence scanning thymine-base analysis has also been introduced. This method provides four thymine-base peak profiles unique for Asian and American/African genotypes of T. solium, T. saginata and T. asiatica. By comparing thymine base peak profiles, it is possible to differentiate human taeniid cestodes without DNA sequencing. The approaches are powerful tools for the routine diagnosis of taeniasis and the molecular identification of taeniid cestodes.


Subject(s)
Animals , Antibodies, Helminth/blood , Antigens, Helminth/genetics , Base Sequence , DNA, Helminth/genetics , Glycoproteins/diagnosis , Humans , Molecular Diagnostic Techniques , Neurocysticercosis/diagnosis , Recombinant Fusion Proteins/diagnosis , Serologic Tests , Taenia/genetics , Taeniasis/diagnosis
6.
Southeast Asian J Trop Med Public Health ; 2002 ; 33 Suppl 3(): 79-82
Article in English | IMSEAR | ID: sea-32733

ABSTRACT

Detection of seven specific bands by immunoblot (IB) using glycoproteins (GPs) purified by lentil-lectin affinity chromatography has been the gold-standard for neurocysticercosis (NCC) serodiagnosis since 1989. However, due to the presence of contaminants, it was impossible to apply the GPs to ELISA. Our group at Asahikawa Medical College (AMC) succeeded in purifying the GPs by preparative isoelectric focusing; these higher quality GPs were suitable for ELISA. Based on the results of both IB and ELISA testing, developed at AMC for a field survey in Irian Jaya, it became evident that that area had pandemic NCC. We found many NCC patients, pigs full of cysts, and one dog infected with two cysts: these findings were based on serology. Recently, we conducted another survey to detect of the worm carriers of T. solium. Three of the 38 local people were positive by copro-antigen specific to Taenia species; these three patients expelled segments of Taenia spp and these were confirmed as those of T. solium by mitochondrial DNA analysis. When viable eggs of any taeniid species could be obtained, they can be developed into metacestodes in NOD-scid mice; it then becomes possible to analyze morphological dynamics, metacestode antigenicity, the efficacy of new metacestocidal drugs, and mitochondrial DNA. Mitochondrial DNA analysis of the specimens obtained in Irian Jaya was compared with that of other isolates worldwide. T. solium is now divided into two genotypes: the Asian type, and the Africa-American type. Some aspects of the pathological differences between the Asian and Africa-American types and the antigenic components of these two types are discussed.


Subject(s)
Animals , Antigens, Helminth/genetics , Asia/epidemiology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Mice, SCID , Polymorphism, Genetic , Serologic Tests , Swine , Taenia solium/immunology , Taeniasis/diagnosis
7.
Southeast Asian J Trop Med Public Health ; 2001 ; 32 Suppl 2(): 105-10
Article in English | IMSEAR | ID: sea-30675

ABSTRACT

DNA vaccine against Cysticercus cellulosae infection was developed and its efficacy was tested. A pair of primers specific to antigen B gene of C. cellulosae was designed which amplified the gene successfully with RT-PCR. The gene was ligated to PV93 vector, and the recombinant of antigen B gene and PV93 was transformed to JM83 cells. The transformed JM83 cells were cultured in a large scale and the plasmid purified. Based on the recombinant plasmid. a DNA vaccine was developed and used to vaccinate two groups of experimental pigs. In each group, there was a routine vaccine, an enhanced vaccine and a control group. Groups 1 and 2 were challenged at 4 months and at 14 days post vaccination respectively with eggs of Taenia solium. The antibody response was also tested with ELISA. The results suggested that all animals vaccinated AgB gene DNA vaccine, no matter by routine or enhanced vaccine, their antibodies reached maximum peak 23 days post vaccination and decreased gradually. When the animals were challenged 4 months after vaccination, they had strong immunity and the parasites decrease rates were 91.2% and 93.1% respectively. When pigs vaccinated with AgB gene DNA vaccine were challenged 14 days post vaccination with 18,000 eggs/pig. The animals showed strong immunity and the parasite decrease rates were 99.5% and 84.9% respectively. However at that time, the antibodies did not reach the peak. While in the control group, the number of C. cellulosae was as many as 2,500. It was concluded that the pigs vaccinated with DNA vaccine had strong immunity against infection of eggs of T. solium.


Subject(s)
Animals , Antibodies, Helminth/biosynthesis , Antigens, Helminth/genetics , Cysticercosis/prevention & control , Cysticercus/genetics , Enzyme-Linked Immunosorbent Assay/veterinary , Meat/parasitology , RNA, Helminth/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Swine , Swine Diseases/parasitology , Treatment Outcome , Vaccination/standards , Vaccines, DNA/administration & dosage , Zoonoses
8.
Article in English | WPRIM | ID: wpr-67266

ABSTRACT

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.


Subject(s)
Amino Acid Sequence , Animals , Antigens, Helminth/genetics , Base Sequence , Cloning, Molecular , Clonorchis sinensis/genetics , DNA, Helminth , Gene Library , Humans , Molecular Sequence Data , Rabbits , Recombinant Proteins , Repetitive Sequences, Nucleic Acid
9.
Southeast Asian J Trop Med Public Health ; 1991 Dec; 22 Suppl(): 256-61
Article in English | IMSEAR | ID: sea-36204

ABSTRACT

Some recently obtained data from our laboratory on the molecular characterization of Echinococcus and Taenia solium are described and are complimented by relevant new information obtained by other groups. Progress made in the development of satisfactory immunodiagnostic assays and in the production of recombinant molecules, suitable for application in serology of hydatid disease and cysticercosis, is highlighted. Results arising from the application of polymerase chain reaction and direct sequencing, using primers homologous to evolutionarily conserved sequences, in phylogenetic studies and for distinguishing individual taeniid species are also discussed.


Subject(s)
Animals , Antibodies, Helminth/blood , Antigens, Helminth/genetics , Cysticercosis/diagnosis , DNA/diagnosis , Echinococcosis/diagnosis , Echinococcus/genetics , Humans , Phylogeny , Recombinant Proteins/immunology , Taenia/genetics , Taeniasis/diagnosis
10.
Article in English | IMSEAR | ID: sea-36259

ABSTRACT

The century-old effort to rid Trichinella spiralis from the food supply has had variable success, and in some regions trichinellosis remains a serious public health concern. However, the research advanced during the past 5 years points toward greater success in developing practical and efficient control strategies. The application of monoclonal antibody and recombinant DNA technologies has permitted great improvement in diagnosis and production of diagnostic reagents. Further, the epidemiology of the disease has undergone considerable revision because of the power of DNA analytical technics which are unravelling the complex genetics of Trichinella spiralis. This improved understanding of the parasite's epidemiology is critical to the design of improved control strategies.


Subject(s)
Animals , Antibodies, Monoclonal/diagnosis , Antigens, Helminth/genetics , Biotechnology , DNA, Recombinant , Humans , Serologic Tests/methods , Trichinella/genetics , Trichinellosis/epidemiology
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