Advantages and limits of real-time PCR assay and PCR-restriction fragment length polymorphism for the identification of cutaneous Leishmania species in Tunisia.

Imène Ben Abda (1, 2) , Frédérique de Monbrison (2) , Nadia Bousslimi (1) , Karim Aoun (1) , Aïda Bouratbine (1) , Stéphane Picot (2)

1 Institut Pasteur de Tunis
2 Service paludisme

Cutaneous leishmaniasis (CL), a public health problem in Tunisia, is associated to three species: Leishmania (L.) infantum, L. major and L. killicki. Accurate and sensitive procedures for the diagnostic of Leishmania infection and for species identification are required to enable adequate treatment and appropriate control measures. Several PCR-methods are applied for the diagnosis and the identification of Leishmania parasites such as PCR-restriction fragment length polymorphism (PCR-RFLP), DNA sequencing, hybridization probes and real-time PCR (RT-PCR). In this study, PCR-RFLP and RT-PCR were performed on skin scrapings from 27 patients with confirmed CL by microscopic examination, in order to compare their usefulness and efficiency for identification of Leishmania species in routine diagnostic laboratories. Identification of Leishmania species was successfully achieved in 96.3% and 81.5% respectively. Agreement between using internal transcribed spacer 1 (ITS1)-PCR-RFLP and kDNA-RT-PCR assays was 70% (19/27). Characterization problems using RT-PCR were mainly due to the difficulties in analyzing the melting temperatures. ITS1-PCR-RFLP and kDNA-RT-PCR presented an interesting alternative to conventional methods for the identification of Leishmania parasites from clinical samples. Both PCR assays can be used in a routine diagnostic, however, further prospective studies including largest sampling, are required to determine their performances in a routine use.

Mots clés: Cutaneous leishmania species; ITS1-PCR-RFLP; kDNA Real-time; PCR; Tunisia

Domaines: Microbiologie et Parasitologie

Imène Ben Abda, Frédérique de Monbrison, Nadia Bousslimi, Karim Aoun, Aïda Bouratbine, et al.. Advantages and limits of real-time PCR assay and PCR-restriction fragment length polymorphism for the identification of cutaneous Leishmania species in Tunisia.. Transactions of The Royal Society of Tropical Medicine and Hygiene, 2011, 105 (1), pp.17-22. ⟨10.1016/j.trstmh.2010.09.003⟩. ⟨pasteur-00683984⟩