Comparative studies of the detection rates of Leishmania parasites from formalin, ethanol-fixed, frozen human skin specimens by polymerase chain reaction and Southern blotting.

ABSTRACT

In this study, detection rates of Leishmania parasites from human skin were compared among three different types of specimens, formalin-fixed, ethanol-fixed, and frozen, by polymerase chain reaction (PCR) and Southern blotting. For this purpose, we used biopsy specimens collected from 19 leishmaniasis patients and performed PCR and Southern hybridization with the probe specific for Leishmania (Viannia) braziliensis complex. Among these 19, 16 specimens were from cutaneous leishmaniasis (CL), one, diffuse cutaneous leishmaniasis (DCL) and 2, mucocutaneous leishmaniasis (MCL) and were formalin-fixed and paraffin-embedded. The causative agents for one case of CL and one case of DCL were already identified as L. (Leishmania) complex. Six specimens of CL were preserved in 100% ethanol. Two specimens of MCL were frozen tissues. PCR using the formalin-fixed and paraffin-embedded specimens revealed positive bands at 70 bp in 9 (47.4%) out of 19 specimens of CL, MCL and DCL. Southern blotting detected the signals in 12 (63.2%) out of the 19. PCR using the 100% ethanol-fixed specimens revealed positive bands in 4 (66.7%) out of 6, and Southern blotting also detected the signals in 4 (66.7%) out of the 6. PCR and Southern blotting using 2 frozen specimens of MCL were always positive (100%). Although we failed to detect significant differences by Chi-square test between the results from the formalin-fixed, paraffin-embedded specimens and those from 100% ethanol-fixed ones, we concluded that ethanol-fixed specimens, convenient for transportation and storage, would be more useful for diagnosis of leishmaniasis by PCR in a developing country.