GENOTYPE CHARACTERIZATION of Leishmania (Viannia) Braziliensis ISOLATED from HUMAN and CANINE BIOPSIES with AMERICAN CUTANEOUS LEISHMANIASIS
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Rev. Inst. Med. Trop. Sao Paulo 57(3):257-262, May-June, 2015 http://dx.doi.org/10.1590/S0036-46652015000300013 GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS Lasaro Teixeira FERREIRA(1), Aparecida Helena de Souza GOMES(2) & Vera Lucia PEREIRA-CHIOCCOLA(1) SUMMARY Introduction: American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo. Methods: Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region ofLeishmania internal transcribed spacers of the ribosomal DNA locus. Results: Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample. Conclusion: These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci. KEYWORDS: American cutaneous leishmaniasis; Leishmania (Viannia) braziliensis; RFLP-PCR; Polymorphism. INTRODUCTION wide geographic distribution1,7,16,18,28. The Leishmania genus causes leishmaniasis, which constitutes a ATL is widely distributed across the Americas. Between 2001 and variety of chronic diseases. There is a wide spectrum of clinical forms, 2011, around 270,500 cases were reported, with an average of 27,500 including those affecting the skin, mucosa, or internal organs16,18. new cases/year. Around 3 - 5% of patients who develop cutaneous lesions are also susceptible to mucosal leishmaniasis23,30. In the state The subgenera Leishmania Viannia is the causative agent of new- of São Paulo there are approximately 400 new cases per year. Another world cutaneous leishmaniasis, comprising the species L. (V.) braziliensis, substantial problem is the urbanization of the infection. Autochthonous L. (V.) panamensis and L. (V.) guyanesis, among others18,26. Infections cases have been reported in urban areas. The incidence of peri-urban and by these species cause three clinical types of American tegumentary urban cases has been increasing. Approximately 10% of the population leishmaniasis (ATL): localized cutaneous, mucosal, and disseminated living in endemic areas is at risk of acquiring the infection29. ATL is also leishmaniasis. Cutaneous lesions are restricted to the entry site of the considered one of the most common dermatological syndromes diagnosed parasites, whereas the mucosal strain is defined by its spreading to the in travelers (or tourists) who have visited endemic areas15. mucosal surfaces of the upper digestive and airway tracts. Disseminated leishmaniasis is characterized by large-scale spreading to distant The life cycle of L. (V.) braziliensis includes different reservoirs, such cutaneous sites2,14,15,24. as humans and wild and domestic mammals, as well as various vector species. Therefore, Leishmania strains can be maintained in both rural and Despite the fact that cutaneous leishmaniasis is caused by at least urban settings, thereby affecting the epidemiology of the infection. Due seven different Leishmania species in Brazil, the vast majority of cases to the proximity of dogs and humans, studies have shown the important are caused by the L. (V.) braziliensis sub-genera, which can be transmitted role of domestic dogs in ATL19,21. Studies using molecular techniques to by different phlebotomine sandfly vectors via animal reservoirs across a characterize L. (V.) braziliensis populations have contributed to a better (1) Laboratório de Biologia Molecular de Parasitas, Instituto Adolfo Lutz, São Paulo, SP, Brazil. (2) Laboratório Regional de Sorocaba, Instituto Adolfo Lutz, Sorocaba, SP, Brazil. Correspondence to: Vera Lucia Pereira-Chioccola, Laboratório de Biologia Molecular de Parasitas, Instituto Adolfo Lutz, Av. Dr Arnaldo 351, 8 andar, 01246-000 São Paulo, SP, Brasil. Phone: +55.11.3068-2991. Fax +55.11.3068-2890. E-mail: [email protected] FERREIRA, L.T.; GOMES, A.H.S. & PEREIRA-CHIOCCOLA, V.L. - Genotype characterization of Leishmania (Viannia) braziliensis isolated from human and canine biopsies with American cutaneous leishmaniasis. Rev. Inst. Med. Trop. Sao Paulo, 57(3): 257-262, 2015. understanding of the abilities of these parasites and their vectors in twice in phosphate-buffered saline (pH 7.2) at 1,000g for 10 min. The adapting to changes in their original forest habitats, and the consequent parasite pellets were used for DNA extraction. L. (V.) braziliensis strain public health implications13. DNA also was used in reactions as a positive control. Despite the significance of ATL to the Brazilian public health DNA purification:Before performing DNA extraction, clinical system, the genetic diversity of L. (V.) braziliensis in some endemic samples and WHO Leishmania reference strains were crushed and areas of Brazil has been poorly researched, as in the state of São Paulo. digested in a lysis buffer until tissue lysis was complete, (This-HCl, Therefore, this study aims to analyze the genetic diversity of a L. (V.) 10 mM, pH 8.0; EDTA 10mM; SDS, 0,5%; N-laurilsarcozil, 0.01%; braziliensis population collected from patients and dogs in the state of proteinase K, 100 µg/mL) by incubation in water bath at 56 °C. Then, São Paulo with cutaneous lesions, avoiding in vitro cultivation. The DNA molecules were extracted by a QIAamp DNA Mini Kit (Qiagen), reason for evaluating polymorphism in humans and dogs was due to the according to the manufacturer’s instructions. DNA concentration and importance of both species within the parasite's life cycle. The results purity was determined by the ratio of O. D. at 260 and 280 nm in a indicate a high variability in isolates collected in patients and dogs from NanoDrop ND1000 (Thermo Scientific). the state of São Paulo. Additionally, this study has shown the possibility of performing genotyping directly on clinical samples without having Routine Leishmania diagnosis to isolate the parasite. Parasitological diagnosis: Skin biopsy imprints were plated onto a MATERIAL AND METHODS glass slide, fixed with methanol and stained with Giemsa11. The presence of amastigotes was observed microscopically with an immersion objective Human and dog samples: The selection of positive samples was (×1,000). made in biopsies received by an in-house Laboratory over a period of nine years (2003 - 2012). The biopsies were collected by medical or PCR targets for Leishmania and internal controls: The Leishmania veterinary health services. The human or canine lesions were cleansed genus was identified by a 120-bp PCR product, amplified from a with antiseptics after the administration of a local anesthetic. The borders conserved region of kDNA minicircles of Leishmania spp., using the of the lesions were scraped or smears of material were obtained by a primer set 150/15223. L. (V.) braziliensis was identified by an amplified punch biopsy of the lesions and immediately added to tubes containing fragment of 146-149 bp from the multicopy spliced leader (SL) RNA 1-2 mL of a sterile 0.85% NaCl and 200 µg/mL gentamicin solution, gene using the primer set LU-5A/LB-3C, which amplifies a 146-149 bp sent to the laboratory within 48 hours and promptly processed to sequence from the SL12,17. These tests were carried out under the same confirm clinical diagnosis. All biopsies recorded were from patients aforementioned conditions11,12. To check PCR inhibitors, canine and human with the cutaneous clinical form. Samples were tested by routine samples were assayed using a reference gene, whose primer sets were diagnosis, which included molecular and parasitological methods. The GAPDH4F/GAPDH4R and β1-β2, respectively, in the same conditions methodologies applied were PCR, using two different sets of primers, and as previously described3,11. After the thermal cycles, PCR products were a parasitological method (microscopic observation). These DNA samples electrophoresed in 2% agarose gel and stained with ethidium bromide. were from patients and dogs living in 36 different municipalities and DNA fragments were made visible under UV illumination. endemic areas for ATL in the state of São Paulo, Brazil (Alumínio, Aruja, Avaré, Bauru, Bragança Paulista, Cajamar, Campinas, Caraguatatuba, L. (V.) braziliensis genotyping by RFLP-PCR (restriction Cerquilho, Conhal, Cubatão, Guapiara, Guarulhos, Ibirá, Ilha Bela, fragment length polymorphism-PCR): Originally, 132 DNA extracts Indaiatuba, Iperó, Iporanga, Itapera, Itupeva, Jaboticabal, Jundiaí, from