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Leishmania sp. identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years

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Abstract

Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.

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Acknowledgements

The authors would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. FAPESP 06/56319-1) and the Laboratório de Patologia de Moléstias Infecciosas (LIM-50/ HC-FMUSP) for their financial support of this project and the Coordenadoria de Aperfeiçoamento Pessoal and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the grant conceded.

Conflicts of interest statement

The authors have no conflicts of interest concerning the work reported in this paper.

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Authors and Affiliations

  1. Pathology Department, Medical School of São Paulo University, São Paulo, Brazil

    Ana Carolina S. de Lima, Thaíse Y. Tomokane, Márcia D. Laurenti, Fernando T. Silveira, Carlos E. P. Corbett & Cláudia M. C. Gomes

  2. Physiology Department, Biosciences Institute of São Paulo University, São Paulo, Brazil

    Ricardo A. Zampieri & Lucile M. Floeter-Winter

  3. Parasitology Department, Evandro Chagas Institute (Surveillance Secretary of Health, Ministry of Health), Belém, Pará, Brazil

    Fernando T. Silveira

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  1. Ana Carolina S. de Lima
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  2. Ricardo A. Zampieri
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  3. Thaíse Y. Tomokane
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  4. Márcia D. Laurenti
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  6. Carlos E. P. Corbett
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  7. Lucile M. Floeter-Winter
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  8. Cláudia M. C. Gomes
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Correspondence to Ana Carolina S. de Lima.

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de Lima, A.C.S., Zampieri, R.A., Tomokane, T.Y. et al. Leishmania sp. identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years. Parasitol Res 108, 1525–1531 (2011). https://doi.org/10.1007/s00436-010-2208-0

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