Leishmaniose visceral canina: avaliação de métodos para detecção de IgG em amostras de soro e eluato

The Brazilian Ministry of Health recommends the culling and euthanasia of dogs with a positive serological test for canine visceral leishmaniasis (CVL). In the Municipality of Rio de Janeiro, the technique used for the diagnosis of CVL is the indirect fluorescent antibody test (IFAT), using blood samples eluted on filter paper (eluate). A dog survey was conducted over a period of one year in the region of Carapiá, in order to evaluate the diagnosis of CVL in this region. All animals underwent clinical examination, and blood samples (serum and eluate) were collected for analysis by enzyme immunoassay (ELISA) and IFAT. A skin biopsy was obtained for parasitological examination (culture). A total of 305 animals were studied and Leishmania chagasi was isolated from nine animals. Sensitivity and specificity were 100% and 96.6% for ELISA, respectively, 100% and 65.5% for IFAT (cut-off at a 1:40 dilution), 100% and 83.4% for IFAT (cut-off at a 1:80 dilution), and 22.2% and 97.0% for eluate IFAT. In conclusion, ELISA was the best tool for the diagnosis of CVL among the serological techniques tested. The present results suggest the need for a better evaluation of filter paper IFAT as the only diagnostic method for CVL in the Municipality of Rio de Janeiro.O Ministério da Saúde recomenda a eutanásia de cães sororreatores como controle da leishmaniose visceral canina (LVC). No Município do Rio de Janeiro, a técnica utilizada para o diagnóstico da LVC é o teste de imunofluorescência indireta (IFI), utilizando amostras de sangue eluídas em papel de filtro (eluato). Um levantamento, durante um ano, foi conduzido na região de Carapiá, a fim de avaliar o diagnóstico da LVC nesta região. Todos os animais foram submetidos a exame clínico e coleta de sangue (soro e eluato) para realização do ensaio imunoenzimático (ELISA) e imunofluorescência indireta (IFI). Biópsia de pele foi obtida para o exame parasitológico (cultura). Foram avaliados 305 (89,4%) animais de uma população de 341 cães e Leishmania chagasi foi isolada de nove animais. A sensibilidade e especificidade do ELISA foram de 100% e 96,6%, na IFI (ponto de corte 1:40) de 100% e 65,5%, na IFI (ponto de corte 1:80) de 100% e 83,4% e na IFI (eluato) de 22,2% e 97,0%, respectivamente. A partir dos resultados obtidos podemos concluir que entre as técnicas sorológicas empregadas, o teste de ELISA apresentou-se como a melhor ferramenta para o diagnóstico da LVC. Os resultados sugerem a necessidade de uma melhor avaliação do teste de IFI realizada com eluato, como único método de diagnóstico para LVC no município do Rio de Janeiro

Comparative assessment of skin reactivity to thimerosal- or phenol-preserved Imunoleish® antigen in dogs with suspected American Tegumentary Leishmaniasis in an endemic area of the state of Rio de Janeiro, Brazil

The leishmanin skin test (LST), which is an in vivo test that assesses the cellular immune responses to Leishmania-derived antigens, is an important tool in the laboratory diagnosis of American tegumentary leishmaniasis (ATL). This study aimed to compare the results obtained in LST employing the Imunoleish® antigen preserved with thimerosal (AgT) or phenol (AgP) and serological techniques to detect a possible infection caused by Leishmania (Viannia) braziliensis in dogs. The study included 172 dogs from an area endemic for ATL in the municipality of Paracambi, state of Rio de Janeiro, Brazil. The results obtained with Imunoleish® antigen preserved with thimerosal (AgT) or phenol (AgP) and serological tests were compared. Each dog received, intradermally, 0.1 mL of each antigen on the inner side of the right (AgT) and left (AgP) thighs. Five (2.7%) dogs presented ATL lesions. Of these, two were reactive to both formulations and three were reactive only to AgT. Among the 172 dogs, 68 (39.5%) were reactive only to AgT, 16 (9.3%)  only to AgP, and 11 (6.4%) to both formulations. Twenty-one (12.2%) sera samples were reactive by immunofluorescent antibody test (IFAT) and 21 enzyme-linked immunosorbent assay (ELISA). However, in only two dogs out of the five which Leishmania was isolated from, serological tests were positive. The LST and serological tests could be a useful tool in the diagnosis of L. (V.) braziliensis infection in dogs. Standardization of the techniques and reagents used could allow comparative studies on sensitivity, specificity, and positive and negative predictive values in dogs from different regions.Keywords: American Tegumentary Leishmaniasis, Leishmanin skin test, Diagnosis, Dogs, Host

Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples

The Brazilian Ministry of Health recommends the culling and euthanasia of dogs with a positive serological test for canine visceral leishmaniasis (CVL). In the Municipality of Rio de Janeiro, the technique used for the diagnosis of CVL is the indirect fluorescent antibody test (IFAT), using blood samples eluted on filter paper (eluate). A dog survey was conducted over a period of one year in the region of Carapiá, in order to evaluate the diagnosis of CVL in this region. All animals underwent clinical examination, and blood samples (serum and eluate) were collected for analysis by enzyme immunoassay (ELISA) and IFAT. A skin biopsy was obtained for parasitological examination (culture). A total of 305 animals were studied and Leishmania chagasi was isolated from nine animals. Sensitivity and specificity were 100% and 96.6% for ELISA, respectively, 100% and 65.5% for IFAT (cut-off at a 1:40 dilution), 100% and 83.4% for IFAT (cut-off at a 1:80 dilution), and 22.2% and 97.0% for eluate IFAT. In conclusion, ELISA was the best tool for the diagnosis of CVL among the serological techniques tested. The present results suggest the need for a better evaluation of filter paper IFAT as the only diagnostic method for CVL in the Municipality of Rio de Janeiro.O Ministério da Saúde recomenda a eutanásia de cães sororreatores como controle da leishmaniose visceral canina (LVC). No Município do Rio de Janeiro, a técnica utilizada para o diagnóstico da LVC é o teste de imunofluorescência indireta (IFI), utilizando amostras de sangue eluídas em papel de filtro (eluato). Um levantamento, durante um ano, foi conduzido na região de Carapiá, a fim de avaliar o diagnóstico da LVC nesta região. Todos os animais foram submetidos a exame clínico e coleta de sangue (soro e eluato) para realização do ensaio imunoenzimático (ELISA) e imunofluorescência indireta (IFI). Biópsia de pele foi obtida para o exame parasitológico (cultura). Foram avaliados 305 (89,4%) animais de uma população de 341 cães e Leishmania chagasi foi isolada de nove animais. A sensibilidade e especificidade do ELISA foram de 100% e 96,6%, na IFI (ponto de corte 1:40) de 100% e 65,5%, na IFI (ponto de corte 1:80) de 100% e 83,4% e na IFI (eluato) de 22,2% e 97,0%, respectivamente. A partir dos resultados obtidos podemos concluir que entre as técnicas sorológicas empregadas, o teste de ELISA apresentou-se como a melhor ferramenta para o diagnóstico da LVC. Os resultados sugerem a necessidade de uma melhor avaliação do teste de IFI realizada com eluato, como único método de diagnóstico para LVC no município do Rio de Janeiro

Characterization of Leishmania spp. causing cutaneous leishmaniasis in Manaus, Amazonas, Brazil

In the State of Amazonas, American tegumentary leishmaniasis is endemic and presents a wide spectrum of clinical variability due to the large diversity of circulating species in the region. Isolates from patients in Manaus and its metropolitan region were characterized using monoclonal antibodies and isoenzymes belonging to four species of the parasite: Leishmania (Viannia) guyanensis, 73% (153/209); Leishmania (Viannia) braziliensis, 14% (30/209); Leishmania (Leishmania) amazonensis, 8% (17/209); and Leishmania (Viannia) naiffii, 4% (9/209). The most prevalent species was L. (V.) guyanensis. The principal finding of this study was the important quantity of infections involving more than one parasite species, representing 14% (29/209) of the total. The findings obtained in this work regarding the parasite are further highlighted by the fact that these isolates were obtained from clinical samples collected from single lesions

PEDIA: prioritization of exome data by image analysis

Purpose Phenotype information is crucial for the interpretation of genomic variants. So far it has only been accessible for bioinformatics workflows after encoding into clinical terms by expert dysmorphologists. Methods Here, we introduce an approach driven by artificial intelligence that uses portrait photographs for the interpretation of clinical exome data. We measured the value added by computer-assisted image analysis to the diagnostic yield on a cohort consisting of 679 individuals with 105 different monogenic disorders. For each case in the cohort we compiled frontal photos, clinical features, and the disease-causing variants, and simulated multiple exomes of different ethnic backgrounds. Results The additional use of similarity scores from computer-assisted analysis of frontal photos improved the top 1 accuracy rate by more than 20–89% and the top 10 accuracy rate by more than 5–99% for the disease-causing gene. Conclusion Image analysis by deep-learning algorithms can be used to quantify the phenotypic similarity (PP4 criterion of the American College of Medical Genetics and Genomics guidelines) and to advance the performance of bioinformatics pipelines for exome analysis

Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines : a case report

Abstract Background Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. Methods Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ+ T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and TH1 polarization. Results A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. Conclusions Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial