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1.
Acta Trop ; 225: 106208, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34687646

RESUMO

The use of serological tests containing multiple immunodominant antigens rather than single antigens have the potential to improve the diagnostic performance in Cystic Echinococcoses (CE) as a complement tool to clear the inconclusive imaging data. Here, we comparatively evaluated the diagnostic value of Hydatid Fluid (HF) and the recently described recombinant multi-epitope antigen DIPOL in IgG-ELISA in a clinically defined cohort of CE patients. The serum samples from 149 CE patients were collected just before surgical or Percutaneous- Aspiration- Injection- Reaspiration (PAIR) procedures. Additionally, serum samples of patients with other parasitic infections (n=49) and healthy individuals (n=21) were also included in the study as controls. To investigate the association between the genotype of the parasite and DIPOL, cyst materials from 20 CE patients were sequenced. In terms of overall sensitivity, HF was higher than DIPOL (82.55%,78.52%, respectively). However, while the sensitivity of HF was higher than DIPOL in patients with active and transitional cysts (83.3%, 75.4%, respectively), sensitivity of DIPOL in inactive cysts was higher compared to HF (95.6%, 78.3%, respectively). The sensitivity of DIPOL depending on cyst stage was statistically significant (P= 0.041). In terms of specificity, DIPOL was found to be better than HF (97.71%, 91.43%, respectively). By genotyping, the majority of 20 patients showed G1 genotype (80%). All patients harboring G3 and G1/G3 cyst genotypes were positive with both antigens, while 87.5% of patients with G1 genotype were seropositive with HF and 75% with DIPOL. The overall sensitivity and high specificity of DIPOL suggest that this recombinant protein containing immunodominant epitopes is a potential substitute for the HF by serological tests for the diagnosis of CE.


Assuntos
Equinococose , Echinococcus granulosus , Animais , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/genética , Equinococose/diagnóstico , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Humanos , Sensibilidade e Especificidade , Testes Sorológicos
2.
PLoS Negl Trop Dis ; 14(11): e0008892, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33253168

RESUMO

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosis and monitoring of CE rely primarily on imaging while serology is used as a confirmatory test. However, imaging is not always conclusive and currently available serological assays have suboptimal sensitivity and specificity, lack standardization, and are not useful for patients´ follow-up. Seroassays for CE are usually based on hydatid fluid (HF), a complex, variable antigenic mixture, and cross-reactivity exists especially with alveolar echinococcosis. Recombinant proteins based on immunogenic antigens most abundant in HF, such as AgB1, AgB2 and Ag5, have been used to overcome these limitations. None of them so far showed potential to replace HF; however, their performance have been largely tested on a limited number of samples, and comparison of different antigens using the same cohort has been rarely performed. The combination of several immunogenic epitopes in a single recombinant protein could enhance test sensitivity. For the diagnosis and follow-up of patients with CE, we compared the performance of the crude HF, previously described recombinant 2B2t antigen, and GST-tagged version of 2B2t, and novel designed recombinants (GST-Ag5t and the GST-DIPOL chimera containing AgB1, AgBB2 and Ag5 epitopes) by IgG-ELISA format. Samples belong to a retrospective cohort of 253 well-characterized patients with CE, previously described for the evaluation of the 2B2t antigen, 92 patients with alveolar echinococcosis, and 82 healthy donors. The reference standard for CE diagnosis was the presence of a CE lesion as diagnosed by ultrasonography. The highest sensitivity was obtained with HF [86.7%, 95% confidence interval (CI): 81.2-91.0], followed by GST-2B2t (70.0%, 95% CI: 63.1-76.2), 2B2t (65.5%, 95% CI: 58.5-72.0), GST-Ag5t (64.5%, 95% CI: 57.5-71.1) and GST-DIPOL (63.1%, 95% CI: 56.0-69.7). The GST-2B2t had the best specificity (95.8%, 95% CI: 88.3-99.1) and the lowest cross-reactivity (38.7%, 95% CI: 27.6-50.6). Good response to treatment also correlated to negative test results in the GST-2B2t ELISA. While none of the tested recombinant antigen appears suitable to replace HF for the diagnosis of CE, GST-2B2t should be further explored as a confirmation test, based on its high specificity and low cross-reactivity, and for the follow-up after treatment in those patients with positive serology for this antigen.


Assuntos
Antígenos de Helmintos/imunologia , Equinococose/diagnóstico , Echinococcus granulosus/imunologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Estudos de Coortes , Reações Cruzadas , Equinococose/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoensaio , Itália , Masculino , Estudos Retrospectivos , Sensibilidade e Especificidade , Testes Sorológicos/métodos
3.
PLoS Negl Trop Dis ; 12(9): e0006741, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30188936

RESUMO

Cystic echinococcosis (CE) is one of the most widespread helminthic zoonoses and is caused by the tapeworm Echinococcus granulosus complex. CE diagnosis and monitoring primarily rely on imaging techniques, complemented by serology. This is usually approached by the detection of IgG antibodies against hydatid fluid (HF), but the use of this heterogeneous antigenic mixture results in a variable percentage of false positive and negative results, and has shown to be useless for follow-up due to the long persistence of anti-HF antibodies in cured patients. To improve test performances and standardization, a number of recombinant antigens mainly derived from HF have been described, among them the B2t and 2B2t antigens. The performance of these antigens in the diagnosis and follow up of patients with CE has been so far evaluated on a limited number of samples. Here, we evaluated the performances of tests based on B2t and 2B2t recombinant antigens compared to HF in IgG-ELISA and immunochromatography (IC) for the diagnosis and follow-up of patients with CE in a retrospective cohort study. A total of 721 serum samples were collected: 587 from 253 patients with CE diagnosed by ultrasonography (US), 42 from patients with alveolar echinococcosis and 92 from healthy donors from Salamanca (Spain). The highest overall sensitivity was obtained with HF in ELISA (85.5%), followed by IC containing HF and 2B2t-HF (83.0% and 78.2%, respectively). The lowest sensitivity was obtained with B2t and 2B2t in ELISA (51.8%). The highest specificity was obtained with IC containing 2B2t-HF (100%), and the lowest with HF-ELISA (78.0%). The lowest cross-reactivity with sera from patients with alveolar echinococcosis was detected with the recombinant antigens in ELISA (9.5% - 16.7%) and the highest with the HF-IC (64.3%). The results of B2t and 2B2t-ELISA were influenced by cyst stage, as classified by US according to the WHO-Informal Working Group on Echinococcosis (WHO-IWGE), with low sensitivity for inactive (CE4 and CE5) cysts, and by the drug treatment, with higher sensitivity in patients after drug treatment compared with patients not subjected to drug treatment. The two recombinant antigens in ELISA provided promising results for monitoring patients in follow-up, although their use is limited to patients with positive serology against them at the beginning of the follow-up. Potential biological reasons behind the low sensitivity of the recombinant antigens and possible strategies to enhance the performance of CE serology are discussed.


Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Equinococose/diagnóstico , Echinococcus granulosus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos de Helmintos/genética , Reações Cruzadas , Feminino , Humanos , Imunoensaio/métodos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/genética , Estudos Retrospectivos , Sensibilidade e Especificidade , Espanha , Adulto Jovem
4.
Vet Parasitol ; 236: 22-33, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-28288760

RESUMO

Cystic echinococcosis (CE) is a chronic and complex zoonotic disease. Information on the mechanisms involved in parasite establishment, growth and persistence remain limited. These may be modulated by a crosstalk between extracellular vesicles (EVs). EVs including exosomes and microvesicles are able to carry developmental signaling proteins which coordinate growth and establishment of several parasites. Here, an exosome enriched EV fraction was isolated from hydatid fluid (HF) of fertile sheep cysts. A proteomic analysis of this fraction identified a number of parasite-derived vesicle-membrane associated proteins as well as cytosolic proteins. Additionally, the exosomal enriched fraction contained proteins of host origin. Specific proteins -antigen B2 and TSPAN14- in the exosomal fraction were further assayed by immunoblot and transmission electron microscopy. To the best of our knowledge, this is the first report on the presence of parasite exosomes in fertile hydatid cyst fluid. Further characterization of the exosome cargo will allow the discovery of new markers for the detection of CE in humans and animals, and the treatment of CE patients, and provide new insights regarding the role of these EVs in the establishment and persistence of hydatid cysts.


Assuntos
Equinococose/veterinária , Echinococcus granulosus/fisiologia , Exossomos/metabolismo , Exossomos/ultraestrutura , Doenças dos Ovinos/patologia , Animais , Equinococose/parasitologia , Equinococose/patologia , Echinococcus granulosus/ultraestrutura , Fertilidade , Immunoblotting/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Proteínas de Protozoários/metabolismo , Ovinos , Doenças dos Ovinos/parasitologia
5.
PLoS Negl Trop Dis ; 10(11): e0005114, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27820824

RESUMO

BACKGROUND: Scientific literature on cystic echinococcosis (CE) reporting data on risk factors is limited and to the best of our knowledge, no global evaluation of human CE risk factors has to date been performed. This systematic review (SR) summarizes available data on statistically relevant potential risk factors (PRFs) associated with human CE. METHODOLOGY/PRINCIPAL FINDINGS: Database searches identified 1,367 papers, of which thirty-seven were eligible for inclusion. Of these, eight and twenty-nine were case-control and cross-sectional studies, respectively. Among the eligible papers, twenty-one were included in the meta-analyses. Pooled odds ratio (OR) were used as a measure of effect and separately analysed for the two study designs. PRFs derived from case-control studies that were significantly associated with higher odds of outcome were "dog free to roam" (OR 5.23; 95% CI 2.45-11.14), "feeding dogs with viscera" (OR 4.69; 95% CI 3.02-7.29), "slaughter at home" (OR 4.67; 95% CI 2.02-10.78) or at "slaughterhouses" (OR 2.7; 95% CI 1.15-6.3), "dog ownership" (OR 3.54; 95% CI 1.27-9.85), "living in rural areas" (OR 1.83; 95% CI 1.16-2.9) and "low income" (OR 1.68; 95% CI 1.02-2.76). Statistically significant PRFs from cross-sectional studies with higher odds of outcome were "age >16 years" (OR 6.08; 95% CI 4.05-9.13), "living in rural areas" (OR 2.26; 95% CI 1.41-3.61), "being female" (OR 1.38; 95% CI 1.06-1.8) and "dog ownership" (OR 1.37; 95% CI 1.01-1.86). CONCLUSIONS/SIGNIFICANCE: Living in endemic rural areas, in which free roaming dogs have access to offal and being a dog-owner, seem to be among the most significant PRFs for acquiring this parasitic infection. Results of data analysed here may contribute to our understanding of the PRFs for CE and may potentially be useful in planning community interventions aimed at controlling CE in endemic areas.


Assuntos
Equinococose/epidemiologia , Equinococose/prevenção & controle , Fatores Etários , Animais , Estudos de Casos e Controles , Estudos Transversais , Bases de Dados Factuais , Cães , Equinococose/parasitologia , Echinococcus/isolamento & purificação , Feminino , Humanos , Fatores de Risco , Fatores Sexuais , Inquéritos e Questionários
6.
J Proteomics ; 136: 145-56, 2016 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-26773860

RESUMO

Parasitic diseases have a great impact in human and animal health. The gold standard for the diagnosis of the majority of parasitic infections is still conventional microscopy, which presents important limitations in terms of sensitivity and specificity and commonly requires highly trained technicians. More accurate molecular-based diagnostic tools are needed for the implementation of early detection, effective treatments and massive screenings with high-throughput capacities. In this respect, sensitive and affordable devices could greatly impact on sustainable control programmes which exist against parasitic diseases, especially in low income settings. Proteomics and nanotechnology approaches are valuable tools for sensing pathogens and host alteration signatures within microfluidic detection platforms. These new devices might provide novel solutions to fight parasitic diseases. Newly described specific parasite derived products with immune-modulatory properties have been postulated as the best candidates for the early and accurate detection of parasitic infections as well as for the blockage of parasite development. This review provides the most recent methodological and technological advances with great potential for bio-sensing parasites in their hosts, showing the newest opportunities offered by modern "-omics" and platforms for parasite detection and control.


Assuntos
Nanotecnologia/métodos , Doenças Parasitárias/diagnóstico , Doenças Parasitárias/metabolismo , Proteômica/métodos , Animais , Humanos
7.
Biomed Res Int ; 2015: 428205, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26504805

RESUMO

Cystic echinococcosis (CE) is an important helminthic zoonotic disease caused by the Echinococcus granulosus complex. In humans, CE is a chronic disease driven by the growth of echinococcal cysts in different organs. Prognosis of this disease depends on multiple factors, including location, number, size, and stage of the cysts, making CE a disease of complex management. CE is usually asymptomatic for years and attracts limited attention from funding organizations and health authorities. For this reason, only experts' recommendations are available but no evidence-based conclusions have been drawn for CE clinical management. One of those pitfalls refers to the lack of evidence to support the use of serological tools for the diagnosis and follow-up of CE patients. In this respect, crude antigens are used to detect specific antibodies in patients, giving rise to false positive results. The advent of molecular techniques allowing the production of recombinant proteins has provided a number of candidate antigens that could overcome the problems associated with the use of crude parasite extracts in the serological assays. In this review, we present the last advances in this field, proposing the use of serology to support cyst stage-specific diagnosis and follow-up.


Assuntos
Equinococose/diagnóstico , Animais , Anticorpos Anti-Helmínticos/análise , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Equinococose/imunologia , Equinococose/parasitologia , Echinococcus granulosus/genética , Echinococcus granulosus/imunologia , Seguimentos , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Humanos , Prognóstico , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Testes Sorológicos/tendências , Zoonoses/diagnóstico , Zoonoses/imunologia , Zoonoses/parasitologia
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