Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.
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.
['de Morais|Rayana Carla Silva|RC|', 'da Costa Oliveira|Cintia Nascimento|CN|', 'de Albuquerque|Suênia da Cunha Gonçalves|Sda C|', 'Mendonça Trajano Silva|Lays Adrianne|LA|', 'Pessoa-E-Silva|Rômulo|R|', 'Alves da Cruz|Heidi Lacerda|HL|', 'de Brito|Maria Edileuza Felinto|ME|', 'de Paiva Cavalcanti|Milena|M|']
D000704:Analysis of Variance D000818:Animals D002415:Cats D016054:DNA, Protozoan D004285:Dogs D006801:Humans D007891:Leishmania D016773:Leishmaniasis, Cutaneous D058885:Multilocus Sequence Typing D012150:Polymorphism, Restriction Fragment Length D060888:Real-Time Polymerase Chain Reaction D044366:Transition Temperature
Species identification Leishmania spp Melting temperature Real time PCR