Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

Abstract

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Key words: Leishmania (V.) braziliensis -diagnosis -polymerase chain reaction -ArgentinaAmerican tegumentary leishmaniasis (cutaneous and mucocutaneous) has been endemic in Argentina since 1916(Mazza 1926. In the Northern provinces of the country, outbreaks were identified in 1985/1987, 1997/1998. In Tucumán province, between 1991and 1996 leishmaniasis cases were reported (Marcolongo et al. 1993, Yadón 1997, Villalonga 1998.In the Americas the causal agents of this pathology are the mexicana complex species: and L. (L.) in the country (Sosa-Estani et al. 1998). At present, the characterization of parasites from human patients in Tucumán has not been possible. Studies from epidemic outbreaks in nearby provinces within Argentina, indicate that the parasites belong to the L. braziliensis complex (Campanini et al. 1993, Cuba et al. 1996, Sosa-Estani et al. 1998. The presence of L. (L.) amazonensis (L. mexicana complex) by isoenzymatic analysis has also been reported in the Chaco region, Argentina (Frank et al. 2000).The most common diagnostic method for cutaneous leishmaniasis used in Argentina is the smear -histopathology exam in combination with clinical and epidemiological data. Mucocutaneous leishmaniasis generally requires a complementary test because of the scarcity of parasites in the lesions.The polymerase chain reaction technique (PCR) has been successfully employed in the detection of specific sequences of pathogenic agents (Erlich 1991). At present this technique is an adequate method for the leishmaniasis diagnosis, showing a high sensitivity when compared to traditional tests (Rodgers et al. 1990, De Bruijn & Barker 1992, De Bruyin et al. 1993, Lopez et al. 1993a, b, Meredith et al. 1993, Piarroux et al. 1994. The sensitivity of the laboratory diagnosis increases significantly when PCR is combined with Southern blotting (Andresen et al. 1996).In the present study we used PCR and Southern blotting for the detection of Leishmania spp. and identification of the parasite from skin biopsies, histologically examined, of human patients from an endemic area of Tucumán province, Argentina. MATERIALS AND METHODSSamples -In this study we used 22 paraffin-embedded skin biopsies from endemic areas of leishmaniasis in the province of Tucumán, kindly donated by the Pathologic Anatomy Service of the Angel C Padilla Hospital (Tucumán, Argentina). Twenty one of the samples had a clinical diagnosis for leishmaniasis and the histopathology test was positive for 13 of them and negative for the rest (Table I). The only sample with a nonleishmanial clinical diagnosis and a negative histopathology result was that used as a negative control.DNA extraction -For each sample five to seven 5 µm sections of paraffin-embedded tissue were placed in a tube. The samples were washed with xilene, incubated for 30 min at 37ºC, centrifuged at 13,000 rpm for 5 min and the supernatant discarded. The washing procedure was repeated three times. Then two washes with eth...

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Urban distribution of Phlebotominae in a cutaneous leishmaniasis focus, Argentina

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Retrospective PCR-based species identification of Leishmania in two patients with visceral leishmaniasis in Serbia

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