Burza S , Croft SL , Boelaert M , 2018. Leishmaniasis. Lancet 392: 951–970.
Antinori S , Giacomelli A & Rezaei N Encyclopedia of Infection and Immunity. New York, NY: Elsevier, 622–643.
Hong A , Zampieri RA , Shaw JJ , Floeter-Winter LM , Laranjeira-Silva MF , 2020. One health approach to leishmaniases: understanding the disease dynamics through diagnostic tools. Pathogens 9: 809.
Veasey JV , Zampieri RA , Lellis RF , Freitas THP , Winter LMF , 2020. Identification of Leishmania species by high-resolution DNA dissociation in cases of American cutaneous leishmaniasis. An Bras Dermatol 95: 459–468.
Aronson N , Herwaldt BL , Libman M , Pearson R , Lopez-Velez R , Weina P , Carvalho EM , Ephros M , Jeronimo S , Magill A , 2016. Diagnosis and treatment of leishmaniasis: clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH). Clin Infect Dis 63: e202–e264.
Reithinger R , Dujardin JC , 2007. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 45: 21–25.
Tokuda Y , Nakamura T , Satonaka K , Maeda S , Doi K , Baba S , Sugiyama T , 1990. Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde. J Clin Pathol 43: 748–751.
Smith LJ , Braylan RC , Nutkis JE , Edmundson KB , Downing JR , Wakeland EK , 1987. Extraction of cellular DNA from human cells and tissues fixed in ethanol. Anal Biochem 160: 135–138.
Wilson IG , 1997. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63: 3741–3751.
Zimmermann J , Hajibabaei M , Blackburn DC , Hanken J , Cantin E , Posfai J , Evans TC Jr , 2008. DNA damage in preserved specimens and tissue samples: a molecular assessment. Front Zool 5: 18.
Greer CE , Lund JK , Manos MM , 1991. PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies. PCR Methods Appl 1: 46–50.
Gillespie JW et al., 2002. Evaluation of non-formalin tissue fixation for molecular profiling studies. Am J Pathol 160: 449–457.
Dahm R , 2005. Friedrich Miescher and the discovery of DNA. Dev Biol 278: 274–288.
Sedlackova T , Repiska G , Celec P , Szemes T , Minarik G , 2013. Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biol Proced Online 15: 5.
Miller SA , Dykes DD , Polesky HF , 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16: 1215.
Uliana SR , Affonso MH , Camargo EP , Floeter-Winter LM , 1991. Leishmania: genus identification based on a specific sequence of the 18S ribosomal RNA sequence. Exp Parasitol 72: 157–163.
Lainson R , Shaw JJ , 1972. Leishmaniasis of the New World: taxonomic problems. Br Med Bull 28: 44–48.
Hammer TJ , Dickerson JC , Fierer N , 2015. Evidence-based recommendations on storing and handling specimens for analyses of insect microbiota. PeerJ 3: e1190.
Franzosa EA et al., 2014. Relating the metatranscriptome and metagenome of the human gut. Proc Natl Acad Sci USA 111: E2329–E2338.
Rotureau B , Gego A , Carme B , 2005. Trypanosomatid protozoa: a simplified DNA isolation procedure. Exp Parasitol 111: 207–209.
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The present work evaluates sampling protocols, storage procedures, and DNA purification methods for Leishmania spp. detection and quantification in different biological samples. The efficiency of three preservation solutions, a phosphate buffer solution, an ethylenediaminetetraacetic acid (EDTA) buffer solution, and 70% ethanol, was compared in combination with three DNA extraction protocols: a commercial silica column kit, salting-out protein precipitation, and organic extraction with phenol-chloroform. Tissue samples from BALB/c mice experimentally infected with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, or Leishmania (Leishmania) infantum were stored in the three preservation solutions and subsequently subjected to the three different DNA extraction methods. The extracted DNA was then used in real-time polymerase chain reaction (PCR) assays for the detection and quantification of parasite ribosomal small subunit DNA targets as well as mammalian glyceraldehyde-3-phosphate dehydrogenase (gapdh) targets. The results of the optimized protocols showed that the DNA extraction method did not influence test quality, but DNA from samples preserved with the EDTA buffer solution produced higher amounts of target amplicons. Based on these results, we concluded that samples from suspected cases of leishmaniasis for submission to molecular diagnostic procedures should be preferentially preserved in EDTA, followed by any one of the DNA purification methods evaluated.
Financial Support: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant nos. 2014/50717-1 and 2018/23512-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico.
Authors’ Addresses: Ricardo Andrade Zampieri, Juliana Ide Aoki, and Lucile Maria Floeter-Winter, Department of Physiology, Institute of Biosciences, University of São Paulo, Sao Paulo, Brazil, E-mails: email@example.com, firstname.lastname@example.org, and email@example.com. Karl Erik Müller, Department of Internal Medicine, Drammen Hospital, Vestre Viken Hospital Trust, Drammen, Norway, and Department of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, Norway, E-mail: firstname.lastname@example.org. Jeffrey Jon Shaw, Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil, E-mail: email@example.com.