Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques

Rayana Carla Silva de Morais, Cintia Nascimento da Costa Oliveira, Suênia da Cunha Gonçalves de Albuquerque, Lays Adrianne Mendonça Trajano Silva, Rômulo Pessoa-e-Silva, Heidi Lacerda Alves da Cruz, Maria Edileuza Felinto de Brito, Milena de Paiva Cavalcanti

Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil.

Abstract

Background

Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmaniaspecies. 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 Leishmaniaspecies identification using two Tm ranges.

Graphical abstract

Keywords: Real time PCR; Melting temperature; Species identification; Leishmania spp.