Angélica Rosa Faria

High-Throughput Analysis of Synthetic Peptides for the Immunodiagnosis of Canine Visceral Leishmaniasis

Background Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis. Methodology/Principal Findings Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs. Conclusions/Significance Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.

Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs

Background Visceral leishmaniasis is the most severe form of leishmaniasis. Worldwide, approximately 20% of zoonotic human visceral leishmaniasis is caused by Leishmania infantum, also known as Leishmania chagasi in Latin America. Current diagnostic methods are not accurate enough to identify Leishmania-infected animals and may compromise the effectiveness of disease control. Therefore, we aimed to produce and test two recombinant multiepitope proteins as a means to improve and increase accuracy in the diagnosis of canine visceral leishmaniasis (CVL). Methodology/Principal Findings Ten antigenic peptides were identified by CVL ELISA in previous work. In the current proposal, the coding sequences of these ten peptides were assembled into a synthetic gene. Furthermore, other twenty peptides were selected from work by our group where good B and T cell epitopes were mapped. The coding sequences of these peptides were also assembled into a synthetic gene. Both genes have been cloned and expressed in Escherichia coli, producing two multiepitope recombinant proteins, PQ10 and PQ20. These antigens have been used in CVL ELISA and were able to identify asymptomatic dogs (80%) more effectively than EIE-LVC kit, produced by Bio-Manguinhos (0%) and DPP kit (10%). Moreover, our recombinant proteins presented an early detection (before PCR) of infected dogs, with positivities ranging from 23% to 65%, depending on the phase of infection in which sera were acquired. Conclusions/Significance Our study shows that ELISA using the multiepitope proteins PQ10 and PQ20 has great potential in early CVL diagnosis. The use of these proteins in other methodologies, such as immunochromatographic tests, could be beneficial mainly for the detection of asymptomatic dogs.

Diagnóstico da Leishmaniose Visceral Canina: grandes avanços tecnológicos e baixa aplicação prática

Visceral leishmaniasis is the most severe type of leishmaniasis, and its zoonotic form, caused by Leishmania (Leishmania) infantum chagasi, accounts for 20% of the reported cases of human leishmaniasis in the world. Its incidence rates are increasing in urban and peri-urban tropical areas. One of the monitoring policies adopted in Brazil is the identification and elimination of infected dogs, which are considered the main reservoir hosts of the disease in cities. Consequently, reliable analyses and diagnoses of these animals are very important for the control of the disease. However, there is not a fast and safe diagnostic method with optimal sensitivity and specificity rates for canine visceral leishmaniasis. This study discussed the advantages and disadvantages of the most common methods available for the evaluation of dogs and the expectations raised by new methods and new antigens for the diagnosis of canine visceral leishmaniasis. Finally, we reviewed the difficulties in the practical applications of these new methodologies.

Immunoreactivity of synthetic peptides derived from proteins of Cryptococcus gattii

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.

Diagnosis of canine visceral leishmaniasis: major technological advances and few practical applications

Visceral leishmaniasis is the most severe type of leishmaniasis, and its zoonotic form, caused by Leishmania (Leishmania) infantum chagasi, accounts for 20% of the reported cases of human leishmaniasis in the world. Its incidence rates are increasing in urban and peri-urban tropical areas. One of the monitoring policies adopted in Brazil is the identification and elimination of infected dogs, which are considered the main reservoir hosts of the disease in cities. Consequently, reliable analyses and diagnoses of these animals are very important for the control of the disease. However, there is not a fast and safe diagnostic method with optimal sensitivity and specificity rates for canine visceral leishmaniasis. This study discussed the advantages and disadvantages of the most common methods available for the evaluation of dogs and the expectations raised by new methods and new antigens for the diagnosis of canine visceral leishmaniasis. Finally, we reviewed the difficulties in the practical applications of these new methodologies.

Novel recombinant multiepitope proteins for the detection of anti-Cryptococcus antibodies

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.

Diagnóstico de la Leishmaniasis Visceral Canina: grandes avances tecnológicos y baja aplicación práctica

Visceral leishmaniasis is the most severe type of leishmaniasis, and its zoonotic form, caused by Leishmania (Leishmania) infantum chagasi, accounts for 20% of the reported cases of human leishmaniasis in the world. Its incidence rates are increasing in urban and peri-urban tropical areas. One of the monitoring policies adopted in Brazil is the identification and elimination of infected dogs, which are considered the main reservoir hosts of the disease in cities. Consequently, reliable analyses and diagnoses of these animals are very important for the control of the disease. However, there is not a fast and safe diagnostic method with optimal sensitivity and specificity rates for canine visceral leishmaniasis. This study discussed the advantages and disadvantages of the most common methods available for the evaluation of dogs and the expectations raised by new methods and new antigens for the diagnosis of canine visceral leishmaniasis. Finally, we reviewed the difficulties in the practical applications of these new methodologies.