Clinical diagnosis of cutaneous leishmaniasis: a comparison study between standardized graded direct microscopy and ITS1-PCR of Giemsa-stained smears.

Parasitological diagnosis of cutaneous leishmaniasis is absolutely necessary before treatment. Direct microscopy of scrapings taken from the margins of skin lesions is the most commonly used method for clinical diagnosis of leishmaniasis. In this study to evaluate the usage of stained smears as samples for PCR and the possible advantage of PCR, we compared the sensitivity of the diagnosis of Giemsa-stained skin scrapings by standardized graded direct microscopy with that of ITS1-PCR with the material of the same area of the slide. Three 5mm x 5mm squares were marked on each of the 20 Giemsa-stained touch smears from 20 clinically diagnosed Palestinian patients. Out of the 60 squares scanned for amastigotes under 100x oil-immersion light microscopy, 45 (75%) gave usable results and 23 of these were positive for Leishmania. Fifteen (25%) squares could not be scanned microscopically, 12 because of staining that was too thick and 3 because of inadequate staining. DNA from each scanned square was extracted separately after microscopy and run through ITS1-PCR. Of the 23 microscopy-positive squares, 20 (87%) of these were positive by PCR. Of the three that were negative, one failed to extract for DNA, the second showed only one amastigote in the entire square, and the third was normally graded as +1 but was not amplified for unknown reasons. Of the 22 squares negative for microscopy, 18 (82%) were ITS1-PCR positive. Additionally, all three improperly stained squares were ITS1-PCR positive. Of the 12 darkly stained squares, 11 were positive. A negative control group of 15 German individuals from which Giemsa-stained slides containing three squares each was prepared and these slides were also microscopically scanned and tested by ITS1-PCR. Both tests were negative with both methods. Compared to microscopy (data in parenthesis), PCR showed a sensitivity of 87% (37%) and a specificity of 100% (100%). We have concluded that Giemsa-stained smears are a readily usable sampling method for PCR and that ITS1-PCR is far more sensitive than microscopy.