High-performance immune diagnosis of tuberculosis: Use of phage display and synthetic peptide in an optimized experimental design.

Immunoassays are practical and cost-effective approaches suitable for large-scale tuberculosis (TB) screening. This study identified new peptide mimotopes of Mycobacterium tuberculosis and applied them in the serodiagnosis of TB. Thereby, linear (X15, X8CX8) and constrained (LX-4 and LX-8) phage display peptide libraries were screened with purified Immunoglobulin G antibodies from TB-positive patients, and eight mimotopes were selected. The mimotope peptides were screened using the SPOT-synthesis technique followed by immunoblotting. Peptides P.Mt.PD.4 and P.Mt.PD.7 demonstrated the highest binding affinity and were chemically synthesized and used as antigens for enzyme-linked immunosorbent assay (ELISA) assays. Experimental designs were used to optimize the assays and to assess each variable's influence. Peptide P.Mt.PD.7 was differentiated between positive and negative samples and achieved 100% sensitivity and specificity when tested on a 100-sera panel. Therefore, the selected peptide was applied to the ELISA assay as a screening method for diagnosing TB represents a potential tool for helping to combat the disease.

Autochthonous canine visceral leishmaniasis cases occur in Paraná state since 2012: isolation and identification of Leishmania infantum / Leishmaniose visceral canina autóctone ocorre no estado do Paraná desde 2012: isolamento e identificação de Leishmania infantum

Abstract The aim of this study was to confirm the emergence of canine visceral leishmaniasis among dogs in Foz do Iguaçu. The disease was diagnosed through the isolation and molecular identification of Leishmania infantum. In the first sample collection stage (2012), three lymph node aspirates and 46 buffy coat samples were obtained mostly from the dogs that were seroreagents for leishmaniasis. In the second sample collection stage (2013), the buffy coat samples were collected from 376 dogs located close to Paraguay, Paraná river, center and peripheral parts of the city. The DNA from the six isolates, four from the first sampling stage (4/49) and two from the second sampling stage (2/376), was subjected to polymerase chain reaction using the K26F/R primers. The isolate was confirmed as L. infantum by sequencing. As none of the dogs had ever left the city, the isolates were confirmed as autochthonous. Further, the study confirmed the emergence of canine visceral leishmaniasis in Paraná through the identification of L. infantum among dogs in Foz do Iguaçu city. Hence, collaborative control measures should be designed and implemented by the public agencies and research institutions of Brazil, Argentina, and Paraguay to control the spread of visceral leishmaniasis.

Resumo O objetivo deste estudo foi confirmar a emergência da leishmaniose visceral canina em Foz do Iguaçu próximo à fronteira com a Argentina e ao Paraguai, por meio do isolamento e identificação molecular de Leishmania infantum. Em um primeiro estágio de coleta de animais (2012), três amostras de aspirados de linfonodos e 46 camadas leucocitárias foram obtidas de cães soropositivos para leishmaniose. Em um segundo estágio de coleta (2013), foram coletadas amostras de camada leucocitária de 376 cães de 20 localidades próximas à fronteira com o Paraguai, rio Paraná, centro e periferia da cidade. Seis isolados foram obtidos, quatro da primeira etapa (4/49) e dois da segunda etapa (2/376); estes isolados foram submetidos à amplificação com iniciadores K26F/R, e a análise de sua sequência confirmou a espécie como L. infantum. A autoctonia dos casos foi confirmada, pois 100% dos cães nunca haviam saído da cidade. O estudo confirma a emergência de leishmaniose visceral canina no Paraná com identificação de L. infantum em cães da cidade de Foz do Iguaçu. Assim, medidas de controle devem ser elaboradas e implementadas por órgãos públicos e instituições de pesquisa do Brasil, Argentina e Paraguai em parceria com o objetivo de controlar a disseminação de zoonoses e os casos humanos de LV.

Autochthonous canine visceral leishmaniasis cases occur in Paraná state since 2012: isolation and identification of Leishmania infantum.

The aim of this study was to confirm the emergence of canine visceral leishmaniasis among dogs in Foz do Iguaçu. The disease was diagnosed through the isolation and molecular identification of Leishmania infantum. In the first sample collection stage (2012), three lymph node aspirates and 46 buffy coat samples were obtained mostly from the dogs that were seroreagents for leishmaniasis. In the second sample collection stage (2013), the buffy coat samples were collected from 376 dogs located close to Paraguay, Paraná river, center and peripheral parts of the city. The DNA from the six isolates, four from the first sampling stage (4/49) and two from the second sampling stage (2/376), was subjected to polymerase chain reaction using the K26F/R primers. The isolate was confirmed as L. infantum by sequencing. As none of the dogs had ever left the city, the isolates were confirmed as autochthonous. Further, the study confirmed the emergence of canine visceral leishmaniasis in Paraná through the identification of L. infantum among dogs in Foz do Iguaçu city. Hence, collaborative control measures should be designed and implemented by the public agencies and research institutions of Brazil, Argentina, and Paraguay to control the spread of visceral leishmaniasis.

Variables associated with the prevalence of anti-Leishmania spp. antibodies in dogs on the tri-border of Foz do Iguaçu, Paraná, Brazil.

The aim of this study was to investigate the occurrence of anti-Leishmania spp. antibodies in dogs from localities in the city of Foz do Iguaçu, Paraná state, Brazil, on the border with Argentina and Paraguay. Blood samples dogs were collected to perform the following serologic tests: immunochromatographic DPP® rapid test, indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence assay (IFA). In 2012, 285 dogs were analyzed on Argentina border, and in 2013, serum samples from 396 dogs on the border of Paraguay were collected. Using ELISA for screening and IFA for the confirmatory test, the results showed that the antibody prevalence was 1.8% (5/285) on the border of Argentina and 3.0% (12/396) on Paraguay border. When using the DPP® for screening and ELISA as a confirmatory analysis, we observed a seroreagent prevalence in dogs of 2.5% (7/285) on Argentina border and 5.1% (20/396) on Paraguay border. The non-public collection of domestic waste (p= 0.0004) was shown to be associated with leishmaniasis. This study shows the presence of leishmaniasis and suggest the emergence of canine visceral leishmaniasis in state of Paraná due to the confirmed occurrence of seroreactive dogs on Argentina and Paraguay border, which has environmental and geographical characteristics that favor the spread of the parasite.

Variables associated with the prevalence of anti-Leishmania spp. antibodies in dogs on the tri-border of Foz do Iguaçu, Paraná, Brazil / Variáveis associadas à prevalência de anticorpos anti- Leishmania spp. em cães na tríplice fronteira de Foz do Iguaçu, Paraná, Brasil

Abstract The aim of this study was to investigate the occurrence of anti-Leishmania spp. antibodies in dogs from localities in the city of Foz do Iguaçu, Paraná state, Brazil, on the border with Argentina and Paraguay. Blood samples dogs were collected to perform the following serologic tests: immunochromatographic DPP® rapid test, indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence assay (IFA). In 2012, 285 dogs were analyzed on Argentina border, and in 2013, serum samples from 396 dogs on the border of Paraguay were collected. Using ELISA for screening and IFA for the confirmatory test, the results showed that the antibody prevalence was 1.8% (5/285) on the border of Argentina and 3.0% (12/396) on Paraguay border. When using the DPP® for screening and ELISA as a confirmatory analysis, we observed a seroreagent prevalence in dogs of 2.5% (7/285) on Argentina border and 5.1% (20/396) on Paraguay border. The non-public collection of domestic waste (p= 0.0004) was shown to be associated with leishmaniasis. This study shows the presence of leishmaniasis and suggest the emergence of canine visceral leishmaniasis in state of Paraná due to the confirmed occurrence of seroreactive dogs on Argentina and Paraguay border, which has environmental and geographical characteristics that favor the spread of the parasite.

Resumo O objetivo deste estudo foi investigar a ocorrência de anticorpos anti- Leishmania spp. em cães da cidade de Foz do Iguaçu, estado do Paraná, Brasil, fronteira com a Argentina e o Paraguai. Amostras de sangue de cães foram coletadas para realização dos seguintes testes sorológicos: teste rápido imunocromatográfico DPP®, ensaio imunoenzimático indireto (ELISA) e ensaio de imunofluorescência indireta (IFI). Em 2012, 285 cães foram analisados na fronteira com Argentina e, em 2013, amostras de soro de 396 cães na fronteira com o Paraguai. Utilizando ELISA para triagem e IFA para o teste confirmatório, os resultados mostraram uma prevalência de anticorpos de 1,8% (5/285) na fronteira da Argentina e 3,0% (12/396) na fronteira com o Paraguai. Ao usar o DPP® para triagem e ELISA como uma análise confirmatória, observou-se uma prevalência de cães sororreagentes de 2,5% (7/285) na fronteira com a Argentina e 5,1% (20/396) na fronteira com o Paraguai. A não coleta pública de lixo doméstico (p = 0,0004) mostrou-se associada à leishmaniose. Este estudo demonstra a presença de leishmaniose e sugere a emergência da leishmaniose visceral canina no estado do Paraná devido à ocorrência de cães sororreagentes na fronteira Argentina e Paraguai, que possuem características ambientais e geográficas que favorecem a disseminação do parasito.

Real-time PCR for traceability and quantification of genetically modified seeds in lots of non-transgenic soybean / PCR em tempo real para rastreabilidade e quantificação de sementes geneticamente modificados em lotes de soja não transgênica

The constant presence of genetically modified (GM) soybean in conventional seed lots has become a growing problem for international seed trade. In this context, seed companies have prompted the development of routine tests for accurate genetically modified soybean seeds detection. In this study, a quantitative PCR-based method was standardized in order to detect and quantify mixtures of seeds (i.e. certified seed) or GM grains (i.e. seeds came from field) into samples of non-GM soybean, in a way that soybean lots can be assessed within the standards established by legislation. The method involved the use of p35S-f2/petu-r1 primers targeting CP-4 enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) gene (i.e. that confers herbicide tolerance in Roundup ReadyTM (RR)) for real-time PCR detection and quantification through mericon Quant GMO Detection Assay. The results revealed the method efficiency to detect and quantify the presence of even one soybean seed in batch used for routine evaluation of GM seeds. In addition, it was possible to detect of up to 0.1% of transgenic DNA relative to the soybean grains content. Thus, the sensitive GMO quantitative approach described in this study will provide support in supervising activities, and facilitate the process and control of GM soybean.

A constante presença da soja geneticamente modificada (GM) em lotes de sementes convencionais têm se tornado um grande problema para o comércio internacional de sementes. Neste contexto, as empresas de sementes estão em busca de testes de rotina extremamente precisos para a detecção de sementes de soja geneticamente modificadas. Neste estudo, um método baseado em PCR quantitativo foi padronizado para detectar e quantificar misturas de sementes (i.e. sementes certificadas) ou grãos geneticamente modificados (i.e. sementes oriundas do campo) dentro de lotes de soja não transgênica, de um modo que os lotes de soja possam ser avaliados dentro dos parâmetros estabelecidos pela legislação. O método envolveu o uso dos iniciadores p35S-f2/petu-r1 alvejando o gene CP-4 5-nolpiruvil-shikimato-3-fosfato sintase (cp4-epsps) (i.e. que confere a tolerância ao herbicida Roundup Ready® (RR)) para detecção e quantificação em PCR de tempo real via Ensaio de detecção Mericon Quant GMO. Os resultados revelaram um método eficiente para detectar e quantificar a presença de até mesmo uma única semente de soja no lote usado para a avaliação de rotina de sementes geneticamente modificadas. Adicionalmente, foi possível detectar até 0,1% de DNA transgênico relativo ao conteúdo de grãos de soja. Dessa forma, uma abordagem quantitativa sensível à soja geneticamente modificada foi descrita nesse estudo e poderá fornecer suporte em atividades de supervisão, além de facilitar o processo de controle da soja geneticamente modificada.

Synthetic Peptides as Potential Antigens for Cutaneous Leishmaniosis Diagnosis.

This work's goal was to research new candidate antigens for cutaneous leishmaniosis (CL). In order to reach the goal, we used random peptide phage display libraries screened using antibodies from Leishmania braziliensis patients. After selection, three peptides (P1, P2, and P3) were synthesized using Fmoc chemistry. The peptides individually or a mixture of them (MIX) was subsequently emulsified in complete and incomplete Freund's adjuvant and injected subcutaneously in golden hamsters. Sera from the hamsters administered with P1 presented antibodies that recognized proteins between 76 and 150 kDa from L. braziliensis. Sera from hamsters which had peptides P2 and P3, as well as the MIX, administered presented antibodies that recognized proteins between 52 and 76 kDa of L. braziliensis. The research on the similarity of the peptides' sequences in protein databases showed that they match a 63 kDa glycoprotein. The three peptides and the MIX were recognized by the sera from CL patients by immunoassay approach (ELISA). The peptides' MIX showed the best performance (79% sensitivity) followed by the P1 (72% sensitivity), and the AS presented 91% sensitivity. These results show a new route for discovering molecules for diagnosis or for immunoprotection against leishmaniosis.

Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy.

BACKGROUND: The diagnosis of leprosy is primarily based on clinical manifestations, and there is no widely available laboratory test for the early detection of this disease, which is caused by Mycobacterium leprae. In fact, early detection and treatment are the key elements to the successful control of leprosy. METHODOLOGY/PRINCIPAL FINDINGS: Peptide ligands for antibodies from leprosy patients were selected from phage-displayed peptide libraries. Three peptide sequences expressed by reactive phage clones were chemically synthesized. Serological assays that used synthetic peptides were evaluated using serum samples from leprosy patients, household contacts (HC) of leprosy patients, tuberculosis patients and endemic controls (EC). A pool of three peptides identified 73.9% (17/23) of multibacillary (MB) leprosy patients using an enzyme-linked immunosorbent assay (ELISA). These peptides also showed some seroreactivities to the HC and EC individuals. The peptides were not reactive to rabbit polyclonal antisera against the different environmental mycobacteria. The same peptides that were conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in the mice. The anti-peptide antibodies that were used in the Western blotting analysis of M. leprae crude extracts revealed a single band of approximately 30 kDa in one-dimensional electrophoresis and four 30 kDa isoforms in the two-dimensional gel. The Western blotting data indicated that the three peptides are derived from the same bacterial protein. CONCLUSIONS/SIGNIFICANCE: These new antigens may be useful in the diagnosis of MB leprosy patients. Their potentials as diagnostic reagents must be more extensively evaluated in future studies using a large panel of positive and negative sera. Furthermore, other test approaches using peptides should be assessed to increase their sensitivity and specificity in detecting leprosy patients. We have revealed evidence in support of phage-displayed peptides as promising biotechnological tools for the design of leprosy diagnostic serological assays.

Rural-urban focus of canine visceral leishmaniosis in the far western region of Santa Catarina State, Brazil.

The aim of this work was to investigate the occurrence of canine visceral leishmaniosis (CVL) in the far western region of Santa Catarina State, bordering Argentina and Parana State, southern Brazil, where in recent years, VL has been recorded in both dogs and humans. Clinical signs, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR) were used for Leishmania investigation. Among the 252 dogs surveyed, 41 were positive by ELISA assay, 43 in IFAT (titer>40), and 55 by PCR. From the 48 positive for VL by both serological and molecular methods, 19 (39.6%) presented clinical symptoms of leishmaniosis, 35 (72.9%) were from rural areas, and 13 (27.1%) were from urban areas. This pilot study confirms the occurrence of VL among dogs in the far western region of Santa Catarina, southern Brazil, with high risk of CVL outbreaks and presenting a threat to humans.

Comparison of conventional serology and PCR methods for the routine diagnosis of Trypanosoma cruzi infection.

INTRODUCTION: Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas disease, and it is estimated that approximately 5 million people in Brazil are infected with this parasite. This work aimed to compare the current diagnostic methods for Chagas disease, including conventional serological (IFAT and ELISA) and molecular techniques (PCR), to introduce PCR as an auxiliary technique. METHODS: A total of 106 chagasic patients were evaluated: 88 from endemic areas of Parana, 6 from São Paulo, 3 from Minas Gerais, 3 from Rio Grande do Sul, 1 from Bahia and 5 from the Santa Catarina T. cruzi outbreak. The samples were analyzed by conventional serological methods (IFAT, ELISA), hemoculture and PCR to confirm Chagas disease. RESULTS: When IFAT was used to determine antibody levels, the sensitivity was 81.7% for patients with the cardiac form of the disease and 100% for the other clinical forms. In contrast, ELISA showed 84% sensitivity and 100% specificity. The use of serological and molecular techniques and their implications for the diagnosis of Chagas disease in non-endemics area are discussed. CONCLUSIONS: PCR constitutes an excellent support methodology for the laboratory diagnosis of Chagas disease due to its high sensitivity and specificity.

Comparison of conventional serology and PCR methods for the routine diagnosis of Trypanosoma cruzi infection

Introduction Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas disease, and it is estimated that approximately 5 million people in Brazil are infected with this parasite. This work aimed to compare the current diagnostic methods for Chagas disease, including conventional serological (IFAT and ELISA) and molecular techniques (PCR), to introduce PCR as an auxiliary technique. Methods A total of 106 chagasic patients were evaluated: 88 from endemic areas of Parana, 6 from São Paulo, 3 from Minas Gerais, 3 from Rio Grande do Sul, 1 from Bahia and 5 from the Santa Catarina T. cruzi outbreak. The samples were analyzed by conventional serological methods (IFAT, ELISA), hemoculture and PCR to confirm Chagas disease. Results When IFAT was used to determine antibody levels, the sensitivity was 81.7% for patients with the cardiac form of the disease and 100% for the other clinical forms. In contrast, ELISA showed 84% sensitivity and 100% specificity. The use of serological and molecular techniques and their implications for the diagnosis of Chagas disease in non-endemics area are discussed. Conclusions PCR constitutes an excellent support methodology for the laboratory diagnosis of Chagas disease due to its high sensitivity and specificity. .

Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

BACKGROUND: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. METHODS: Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. RESULTS: Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. CONCLUSIONS: The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

PCR-restriction fragment length polymorphism analysis as a tool for Mycobacterium species identification in lepromas for lepromin production.

OBJECTIVES: The aim of the present work was to standardise a PCR-Restriction Fragment Length Polymorphism analysis (PRA) as a tool to detect the mycobacteriologic composition of lepromas from leprosy patients used in the production of lepromin to improve the quality of the Mitsuda test. DESIGN: PCR-Restriction Fragment Length Polymorphism analysis using hsp65 and rpoB genes were applied to 11 reference strains of mycobacteria, including M. leprae, and the obtained PRA profiles were compared to mycobacteria in clinical specimens. RESULTS: Out of the biopsies studied, 522% had DNA fragment amplified for both genes (hsp65 and rpoB) for M. leprae. However, other Mycobacterium species were observed in samples of lepromatous leprosy patients. Here we discussed the importance of mycobacteria identification in the antigen of Mitsuda production to be used in the evaluation of leprosy. CONCLUSIONS: Our results suggest that the use of the molecular approach for sample selection can contribute to an improvement in the quality of produced lepromin.