title: Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.
abstract: BACKGROUND
  Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques.
  
  
  METHODS
  qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains.
  
  
  RESULTS
  Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing and qPCR (p = 0.2566). In contrast, a highly significant difference was observed for qPCR and RFLP (p < 0.001).
  
  
  CONCLUSIONS
  In this study, we demonstrated the potential use of qPCR as a tool for Leishmania species identification using two Tm ranges.
authors.affiliation: Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: rayanacarla_m@hotmail.com.
authors.forename: Rayana Carla Silva
authors.initials: RC
authors.lastname: de Morais
authors.affiliation: Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: cintiancosta_91@hotmail.com.
authors.forename: Cintia Nascimento
authors.initials: CN
authors.lastname: da Costa Oliveira
authors.affiliation: Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: sueniaaa@hotmail.com.
authors.forename: Suênia da Cunha Gonçalves
authors.initials: Sda C
authors.lastname: de Albuquerque
authors.affiliation: Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: laystrajano@gmail.com.
authors.forename: Lays Adrianne
authors.initials: LA
authors.lastname: Mendonça Trajano Silva
authors.affiliation: Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: romulops12@gmail.com.
authors.forename: Rômulo
authors.initials: R
authors.lastname: Pessoa-E-Silva
authors.affiliation: Federal University of Pernambuco, Cidade Universitária, Av. Moraes Rego s/n, CEP 50670-420, Recife-PE, Brazil. Electronic address: heidi.alves@gmail.com.
authors.forename: Heidi Lacerda
authors.initials: HL
authors.lastname: Alves da Cruz
authors.affiliation: Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: britomef@cpqam.fiocruz.br.
authors.forename: Maria Edileuza Felinto
authors.initials: ME
authors.lastname: de Brito
authors.affiliation: Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: mp@cpqam.fiocruz.br.
authors.forename: Milena
authors.initials: M
authors.lastname: de Paiva Cavalcanti
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002415:Cats; D016054:DNA, Protozoan / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D004285:Dogs; D006801:Humans; D007891:Leishmania / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis / Q000469:parasitology*; D058885:Multilocus Sequence Typing; D012150:Polymorphism, Restriction Fragment Length; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D044366:Transition Temperature
chemical_list: D016054:DNA, Protozoan
keywords: Leishmania spp; Melting temperature; Real time PCR; Species identification