Detection of Internal Transcribed Spacer 1 and hsp70 Genetic Markers Using Restriction Fragment Length Polymorphisms and Sequencing in Identification of Leishmania Species Causing Tegumentary Leishmaniasis in Brazil

Jaqueline Alves Delprete Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil;
Instituto de Infectologia Emílio Ribas, São Paulo, Brazil;

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Livia Vieira de Almeida Instituto de Infectologia Emílio Ribas, São Paulo, Brazil;

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Alessandra Moraes Barros Instituto de Infectologia Emílio Ribas, São Paulo, Brazil;

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Rita de Cássia Soler Instituto de Infectologia Emílio Ribas, São Paulo, Brazil;

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Amanda Azevedo Bittencourt Instituto de Infectologia Emílio Ribas, São Paulo, Brazil;

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Expedito José de Albuquerque Luna Departamento de Medicina Preventiva, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil;

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José Angelo Lauletta Lindoso Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil;
Instituto de Infectologia Emílio Ribas, São Paulo, Brazil;
Laboratório de Protozoologia (LIM-56 HC-FMUSP), Instituto de Medicina Tropical, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil

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Lúcia Maria Almeida Braz Departamento de Medicina Preventiva, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil;

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ABSTRACT.

The identification of Leishmania species that cause tegumentary leishmaniasis (TL) is important for taxonomic and prognostic purposes. Molecular analysis using different Leishmania genomic targets is the most useful method for identifying Leishmania species. Therefore, we evaluated the performance of ribosomal RNA internal transcribed spacer 1 (ITS1) and heat shock protein (hsp70) genetic markers by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis (RFLP) and sequencing, for identification of Leishmania species. Samples from 84 Brazilian patients were amplified. Internal transcribed spacer 1 PCR followed by RFLP (HaeIII) [ITS1-RFLP (HaeIII)] identified 46.4% (39/84) of the samples as compatible with the Viannia subgenus. Internal transcribed spacer 1 PCR followed by sequencing (ITS1-sequencing) identified Leishmania (Viannia) braziliensis in 91.7% (77/84) of the TL samples, Leishmania (Leishmania) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 2.4% (2/84), and L. (L.) infantum in 1.2% (1/84). One of the samples showed the same proportion of similarity with L. (V.) guyanensis and L. (V.) panamensis. hsp70 nested PCR followed by RFLP (HaeIII) [nested hsp70-RFLP (HaeIII)] identified 91.7% (77/84) of the samples as compatible with L. (V.) braziliensis/L. (V.) naiffi, 3.6% (3/84) with L. (L.) amazonensis, 1.2% (1/84) with L. (L.) infantum, and 3.6% (3/84) with L. (V.) guyanensis. hsp70 PCR followed by sequencing (hsp70-sequencing) identified L. (V.) braziliensis in 91.7% (77/84) of the TL samples, L. (L.) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 3.6% (3/84), and L. (L.) infantum in 1.2% (1/84). Our findings clearly showed that nested hsp70-RFLP (HaeIII) is better than ITS1-RFLP (HaeIII) and that ITS1 or hsp70 PCR followed by sequencing was adequate for identifying Leishmania species. We also found that Leishmania (Viannia) braziliensis is the most common species causing TL in Brazil. Therefore, sequencing multiple target genes such as ITS1 and hsp 70 is more accurate than RFLP for identifying Leishmania species.

Author Notes

Financial support: This study was funded by grant 19375-0/2019 from the São Paulo Research Foundation (FAPESP).

Authors’ addresses: Jaqueline Alves Delprete, Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil, and Instituto de Infectologia Emílio Ribas, São Paulo, Brazil, E-mail: delprete@usp.br. Livia Vieira de Almeida, Alessandra Moraes Barros, Rita de Cássia Soler, and Amanda Azevedo Bittencourt, Instituto de Infectologia Emílio Ribas, São Paulo, Brazil, E-mails: livia.almeida@emilioribas.sp.gov.br, alebarros.derma@gmail.com, rita.soler@emilioribas.sp.gov.br, and am.bitten@gmail.com. Expedito José de Albuquerque Luna and Lúcia Maria Almeida Braz, Departamento de Medicina Preventiva, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil, E-mails: eluna@usp.br and lmabraz@usp.br. José Angelo Lauletta Lindoso, Departamento de Doenças Infecciosas e Parasitárias, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil, Instituto de Infectologia Emílio Ribas, São Paulo, Brazil, and Laboratório de Protozoologia (LIM-56 HC-FMUSP), Instituto de Medicina Tropical, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, E-mail: jlindoso@usp.br.

Address correspondence to Lúcia Maria Almeida Braz, Departamento de Medicina Preventiva, Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Enéas Carvalho de Aguiar, 470, São Paulo, SP CEP 05403900, Brazil. E-mail: lmabraz@usp.br
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