Rayana Carla Silva de Morais1, Cintia Nascimento da Costa Oliveira2, Suênia da Cunha Gonçalves de Albuquerque3, Lays Adrianne Mendonça Trajano Silva4, Rômulo Pessoa-E-Silva5, Heidi Lacerda Alves da Cruz6, Maria Edileuza Felinto de Brito7, Milena de Paiva Cavalcanti8. 1. Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: rayanacarla_m@hotmail.com. 2. Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: cintiancosta_91@hotmail.com. 3. Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: sueniaaa@hotmail.com. 4. Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: laystrajano@gmail.com. 5. Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: romulops12@gmail.com. 6. Federal University of Pernambuco, Cidade Universitária, Av. Moraes Rego s/n, CEP 50670-420, Recife-PE, Brazil. Electronic address: heidi.alves@gmail.com. 7. Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: britomef@cpqam.fiocruz.br. 8. Aggeu Magalhães Research Center, Cidade Universitária, Av. Moraes Rego, CEP 50670-420, Recife-PE, Brazil. Electronic address: mp@cpqam.fiocruz.br.
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.
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: Yun Wu; Xiaojun Tian; Nan Song; Minjun Huang; Zhaoyong Wu; Shaogang Li; Nicholas R Waterfield; Bin Zhan; Lei Wang; Guowei Yang Journal: Front Cell Infect Microbiol Date: 2020-10-07 Impact factor: 5.293