title: American tegumentary leishmaniasis: effectiveness of an immunohistochemical protocol for the detection of Leishmania in skin.
abstract: BACKGROUND
  American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody.
  
  
  METHODS
  Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples.
  
  
  RESULTS
  The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system).
  
  
  CONCLUSIONS
  The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.
authors.affiliation: Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil.
authors.forename: Cibele Fontes
authors.initials: CF
authors.lastname: Alves
authors.forename: Cintia Fontes
authors.initials: CF
authors.lastname: Alves
authors.forename: Maria Marta
authors.initials: MM
authors.lastname: Figueiredo
authors.forename: Carolina Carvalho
authors.initials: CC
authors.lastname: Souza
authors.forename: George Luiz Lins
authors.initials: GL
authors.lastname: Machado-Coelho
authors.forename: Maria Norma
authors.initials: MN
authors.lastname: Melo
authors.forename: Washington Luiz
authors.initials: WL
authors.lastname: Tafuri
authors.forename: Pedro
authors.initials: P
authors.lastname: Raso
authors.forename: Rodrigo Pedro
authors.initials: RP
authors.lastname: Soares
authors.forename: Wagner Luiz
authors.initials: WL
authors.lastname: Tafuri
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001938:Brazil; D002648:Child; D002675:Child, Preschool; D004285:Dogs; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D007891:Leishmania / Q000302:isolation & purification*; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology* / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D018512:Parasitemia / Q000469:parasitology / Q000473:pathology; D012867:Skin / Q000469:parasitology* / Q000473:pathology*; D055815:Young Adult