Anais Brasileiros de Dermatologia
On-line version ISSN 1806-4841
GARCIA, Flávio C. Barbosa et al. Subsidiary methods for the diagnosis of American tegumentar leishmaniasis (ATL): comparison of sequencing of DNA and PCR-RFLP for identification of leishmania species in skin samples. An. Bras. Dermatol. [online]. 2005, vol.80, suppl.3, pp.S339-S344. ISSN 1806-4841. http://dx.doi.org/10.1590/S0365-05962005001000013.
BACKGROUND: ATL is endemic in Brazil, and molecular methods have been shown more effective for its diagnosis. OBJECTIVES: Our purpose was to compare the results of Montenegros skin reaction (MR), presence of leishmania in skin biopsy (Bx), indirect immunofluorescence (IIF) for leishmania in sera samples, PCR, sequencing of DNA and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) as subsidiary exams for ATL diagnose. METHODS: 152 patients were studied, with accomplishment of MR, Bx, IIF and PCR for leishmania in skin samples. For PCR, a specific pair of primers for a sequence of 120bp from kDNA minicircle, common to all leishmanias species, was used. The product of PCR was used for DNA sequencing and for RFLP with the enzyme Hae III. The analysis of the restriction pattern was compared to the cultures of L. (L.) amazonensis and L. (V.) braziliensis. RESULTS: The predominant sex was male (75%), the white color (80%) and urban professional occupation (48%). The age varied from 3 to 77 years, with prevalence from 21 to 50 years (56.5%). In relation to the origin, 65.8% was from the state of São Paulo, being the cutaneous form the more prevalent (79.6%). MR was positive in 73.4% and there was presence of leishmania in 30.6% of the samples, while IIF presented 59.7% of positivity. PCR was positive in 81.6% of skin samples, and PCR-RFLP identified L. (V.) braziliensis (66%) as predominant species, fact that also happened with DNA sequencing, with 64.4% of the positive results for L. (V.) braziliensis. Comparing PCR-RFLP and DNA sequencing there was 61% of agreement amongst the results, being significant in PCR-RFLP for L. (V.) braziliensis. CONCLUSION: MR and PCR were equivalent as subsidiary methods for the diagnosis of ATL, such as PCR-RFLP and DNA sequencing in the identification of the leishmania species. On the other hand, the method PCR-RFLP presented smaller cost and smaller time of execution compared to the sequencing of DNA.
Keywords : DNA; Leishmaniasis; Polymerase chain reaction.