Listeria monocytogenes: review of pathogenesis and virulence determinants-targeted immunological assays.

Listeria monocytogenes is one of the most invasive foodborne pathogens and is responsible for numerous outbreaks worldwide. Most of the methods to detect this bacterium in food require selective enrichment using traditional bacterial culture techniques that can be time-consuming and labour-intensive. Moreover, molecular methods are expensive and need specific technical knowledge. In contrast, immunological approaches are faster, simpler, and user-friendly alternatives and have been developed for the detection of L. monocytogenes in food, environmental, and clinical samples. These techniques are dependent on the constitutive expression of L. monocytogenes antigens and the specificity of the antibodies used. Here, updated knowledge on pathogenesis and the key immunogenic virulence determinants of L. monocytogenes that are used for the generation of monoclonal and polyclonal antibodies for the serological assay development are summarised. In addition, immunological approaches based on enzyme-linked immunosorbent assay, immunofluorescence, lateral flow immunochromatographic assays, and immunosensors with relevant improvements are highlighted. Though the sensitivity and specificity of the assays were improved significantly, methods still face many challenges that require further validation before use.

Alanine Substitution Inactivates Cross-Reacting Epitopes in Dengue Virus Recombinant Envelope Proteins.

The expansion of the habitat of mosquitoes belonging to the Aedes genus puts nearly half of the world's population at risk of contracting dengue fever, and a significant fraction will develop its serious hemorrhagic complication, which can be fatal if not diagnosed properly and treated in a timely fashion. Although several diagnostic methods have been approved for dengue diagnostics, their applicability is limited in rural areas of developing countries by sample preparation costs and methodological requirements, as well as cross-reactivity among the different serotypes of the Dengue virus and other flavivirus, such as the Zika virus. For these reasons, it is necessary to generate more specific antigens to improve serological methods that could be cheaper and used in field operations. Here, we describe a strategy for the inactivation of cross-reacting epitopes on the surface of the Dengue virus envelope protein through the synthetic generation of recombinant peptide sequences, where key amino acid residues from Dengue virus serotype 1 (DENV-1) and 2 (DENV-2) are substituted by alanine residues. The proteins thus generated are recognized by 88% of sera from Dengue NS1+ patients and show improved serotype specificity because they do not react with the antibodies present in seroconverted, PCR-serotyped DEN-4 infected patients.

Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis.

An accurate diagnosis of visceral leishmaniasis is an essential tool for control of the disease. While serologic methods are very useful, these conventional methodologies still present limitations in terms of sensitivity and specificity. The use of flow cytometry is a worldwide trend in the development of high-performance diagnostic methods. Herein, we describe a new flow cytometry serology test, characterized by the employment of the Cytometric Bead Array microspheres A4 and E4 coated with the recombinant antigens rLci1A and rLci2B respectively, to improve the serodiagnosis of canine visceral leishmaniasis. The tests were conducted in a wide variety of sera groups (n = 140), where the diagnostics development would be optimized accounting not just the ability to identify infected dogs with different clinical status, but also to exclude cross-reaction and differentiate vaccinated dogs from dogs infected. Serological testing of the antigenic system A4-rLci1A showed a sensitivity of 90.0% and specificity of 75%, while the E4-rLci2B testing demonstrated a sensitivity of 95.0% and specificity of 82.5%. The use of a multiplex assay of A4-rLci1A and E4-rLci2B, resulted in a diagnostic improvement, with a sensitivity of 95.0% and specificity of 91.2%. Our results show that this novel flow cytometry serology test is a viable tool for sensitive and specific serodiagnosis. Notably, the combination of distinct antigenic systems allows us to test for antibodies to multiple recombinant antigens from a single serum sample. This benefit emphasizes the importance of this methodology as an alternative in the serological diagnosis.

Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples / 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 percent and 96.6 percent for ELISA, respectively, 100 percent and 65.5 percent for IFAT (cut-off at a 1:40 dilution), 100 percent and 83.4 percent for IFAT (cut-off at a 1:80 dilution), and 22.2 percent and 97.0 percent 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 por cento) animais de uma população de 341 cães e Leishmania chagasi foi isolada de nove animais. A sensibilidade e especificidade do ELISA foram de 100 por cento e 96,6 por cento, na IFI (ponto de corte 1:40) de 100 por cento e 65,5 por cento, na IFI (ponto de corte 1:80) de 100 por cento e 83,4 por cento e na IFI (eluato) de 22,2 por cento e 97,0 por cento, 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.