title: Quantitative real time PCR assays for the detection of Leishmania (Viannia) braziliensis in animals and humans.
abstract: American cutaneous leishmaniasis (ACL) caused by Leishmania (Viannia) braziliensis is a neglected disease of humans in the New World that may also cause irreversible skin and eventually mucocutaneous lesions. This parasite can also infect dogs and represents a diagnostic challenge for veterinarians. Methods currently available for the diagnosis of ACL have a low sensitivity and may be time-consuming, representing a limit for treatment expedition of ACL. Quantitative real time PCR assays (qPCR) for the detection of L. (V.) braziliensis in canine blood samples were developed herein, and the detection limit and specificity of different molecular targets (kDNA and rDNA) evaluated. Of the protocols assessed, two qPCR assays, one targeting the kDNA and other the SSU rDNA of L. (V.) braziliensis, performed better, with detection limits of 100 fg and 10 pg, respectively. These assays were also used to test skin samples from humans with suspected ACL. The results indicate that the qPCR protocols developed represent an advance for the diagnosis of ACL in dogs and humans from this region, and provide a rapid and non-invasive diagnosis of the infection by L. (V.) braziliensis. Considering the quantitative nature of the assays, they will also be useful for monitoring treatment efficacy and preventing relapses in human patients in Brazil, although further studies are needed to critically evaluate the specificity of the qPCRs for their capacity to distinguish different Leishmania species and subspecies (represented by zymodemes) in other countries. Finally, molecular assays established may represent new tools for future basic and applied research focused on species identification, host-parasite associations, and infection dynamics in host and vector populations.
authors: Department of Immunology, Aggeu Magalhães Research Centre, Oswaldo Cruz Foundation, 50670-420 Recife, Pernambuco, Brazil. mp@cpqam.fiocruz
authors: Milena
authors: M
authors: de Paiva Cavalcanti
authors: Filipe
authors: F
authors: Dantas-Torres
authors: Suênia
authors: S
authors: da Cunha Gonçalves de Albuquerque
authors: Rayana Carla
authors: RC
authors: Silva de Morais
authors: Maria Edileuza Felinto
authors: ME
authors: de Brito
authors: Domenico
authors: D
authors: Otranto
authors: Sinval Pinto
authors: SP
authors: Brandão-Filho
mesh_terms: D000818:Animals; D004275:DNA, Ribosomal / Q000737:chemistry; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D006801:Humans; D007892:Leishmania braziliensis / Q000145:classification / Q000235:genetics*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology / Q000662:veterinary*; D058069:Neglected Diseases; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004275:DNA, Ribosomal