Comparison of paraffin-embedded skin biopsies from different anatomical regions as sampling methods for detection of Leishmania infection in dogs using histological, immunohistochemical and PCR methods

BACKGROUND: We compared skin biopsy samples from different anatomical regions for detecting Leishmania in dogs, using histological (HE), immunohistochemical (IHC) and polymerase chain reaction (PCR) techniques. RESULTS: The sensitivity was 82.8 percent for PCR, 62.1 percent for IHC and 44.8 percent for HE. These methods do not appear to depend on the clinical status of the animal or the anatomical source of the skin sample; there is no "best region" for any method. However, PCR was more effective than IHC and HE for ear and nose skin samples whereas IHC was better than HE for nose samples. There was weak agreement between PCR and HE for all tissue samples; good agreement between PCR and IHC for ear and abdomen samples, and weak agreement for nose; and optimal agreement between IHC and HE for ear and abdomen and good agreement for nose samples. CONCLUSION: The PCR on ear skin could be the best procedure for diagnosing canine visceral leishmaniasis. The good agreement between PCR and IHC indicates that IHC can be used as an alternative method. Finally, tissue samples from ears, nose and abdomen, particularly ears and nose, are potentially useful for diagnosing canine visceral leishmaniasis independently of the animal's clinical status

Processo e kit para imunodiagnóstico da criptococose utilizando peptídeos sintéticos imunorreativos

Universidade Federal do Rio Grande do SulUniversidade Federal de Minas GeraisUniversidade Federal do PiauíCiências BiológicasDepositad

Use of PCR–RFLP to identify Leishmania species in naturally-infected dogs.

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania ( Leishmania ) chagasi and Leishmania ( Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive ( 1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic ( n = 12) and asymptomatic ( n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. ( L.) chagasiexcept one, infected withL. ( V.) braziliensis . PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR–RFLP can be used to identify Leishmania species in dog tissue imprint stained slides

Visible LED light driven photoelectroanalytical detection of antibodies of visceral leishmaniasis based on electrodeposited CdS film sensitized with Au nanoparticles

FAPEMA - FUNDAÇÃO DE AMPARO A PESQUISA E AO DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO DO MARANHÃOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOThis work describes the development of a high sensitive photoelectrochemical immunosensor for the detection of anti-Leishmania infantum antibodies. The proposed sensor is based on cadmium sulfide films and gold nanoparticles deposited on indium tin oxide256682690FAPEMA - FUNDAÇÃO DE AMPARO A PESQUISA E AO DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO DO MARANHÃOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPEMA - FUNDAÇÃO DE AMPARO A PESQUISA E AO DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO DO MARANHÃOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOPRONEM-00155/1600927/16UNIVERSAL-01194/17303525/2016-9421139/2016-1305680/2015-3426337/2016-6The authors are grateful to FAPEMA (PRONEM-00155/16 ; UNIVERSAL-00927/16 ; UNIVERSAL-01194/17), CNPq (303525/2016-9 ; 421139/2016-1 ; 305680/2015-3 ; 426337/2016-6), and Instituto Nacional de Ciência e Tecnologia em Bioanalítica (465389/2014-7) for

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

Immuno-biochemical evaluations of phenol and thimerosal as antigen preservatives in Montenegro skin test.

Montenegro skin test (MST) represents the main complementary diagnostic test for tegumentary leishmaniases (TL) in endemic regions. Most antigen formulations used for the MST contain thimerosal as preservative. The Food and Drug Administration (FDA), however, recommended reducing or eliminating thimerosal from vaccines and other biological reagents and the Agˆencia Nacional de Vigilˆancia Sanit´aria (ANVISA) in Brazil, prohibited the use of mercurial compounds in immunobiologicals. In the search for an alternative stabilizer, phenol and thimerosal were tested as antigen preservatives in MST. Formulations were tested when fresh and after a 12-month storage at 4 ◦C in TL confirmed mice and human patients, and were evaluated for protein constitution by SDS-PAGE, Western blot and anti-gp63 ELISA. In mice, a decrease in the diagnostic effectiveness in merthiolate formulation was observed after a 12-month storage. SDS-PAGE, Western blot and anti-gp63 ELISA analyses showed a degradation of antigen proteins in both formulations after 12-month storage and that phenol-preserved antigen was quantitatively and qualitatively better than the merthiolate-preserved one. In patients, the average of induration diameter was larger in fresh antigens (p < 0.05). However, storage time did not jeopardize their diagnostic capacity.No non-specific reactions produced by phenol or merthiolate were observed neither in humans nor in mice. Phenol could be a good alternative to replace the merthiolate in MST, and despite the proteolytic activity, antigens remain viable for at least 12 months

Immuno-biochemical evaluations of phenol and thimerosal as antigen preservatives in Montenegro skin test.

Submitted by Karine Ribeiro ([email protected]) on 2014-10-23T16:18:44Z No. of bitstreams: 1 ARTIGO_ImmunoBiochemicalEvaluations.pdf: 169723 bytes, checksum: df4891a1400612d3ee39a7e22e1828bb (MD5)Approved for entry into archive by Gracilene Carvalho ([email protected]) on 2014-11-11T18:23:47Z (GMT) No. of bitstreams: 1 ARTIGO_ImmunoBiochemicalEvaluations.pdf: 169723 bytes, checksum: df4891a1400612d3ee39a7e22e1828bb (MD5)Made available in DSpace on 2014-11-11T18:23:47Z (GMT). No. of bitstreams: 1 ARTIGO_ImmunoBiochemicalEvaluations.pdf: 169723 bytes, checksum: df4891a1400612d3ee39a7e22e1828bb (MD5) Previous issue date: 2006Montenegro skin test (MST) represents the main complementary diagnostic test for tegumentary leishmaniases (TL) in endemic regions. Most antigen formulations used for the MST contain thimerosal as preservative. The Food and Drug Administration (FDA), however, recommended reducing or eliminating thimerosal from vaccines and other biological reagents and the Agˆencia Nacional de Vigilˆancia Sanit´aria (ANVISA) in Brazil, prohibited the use of mercurial compounds in immunobiologicals. In the search for an alternative stabilizer, phenol and thimerosal were tested as antigen preservatives in MST. Formulations were tested when fresh and after a 12-month storage at 4 ◦C in TL confirmed mice and human patients, and were evaluated for protein constitution by SDS-PAGE, Western blot and anti-gp63 ELISA. In mice, a decrease in the diagnostic effectiveness in merthiolate formulation was observed after a 12-month storage. SDS-PAGE, Western blot and anti-gp63 ELISA analyses showed a degradation of antigen proteins in both formulations after 12-month storage and that phenol-preserved antigen was quantitatively and qualitatively better than the merthiolate-preserved one. In patients, the average of induration diameter was larger in fresh antigens (p < 0.05). However, storage time did not jeopardize their diagnostic capacity.No non-specific reactions produced by phenol or merthiolate were observed neither in humans nor in mice. Phenol could be a good alternative to replace the merthiolate in MST, and despite the proteolytic activity, antigens remain viable for at least 12 months