PCR Detection and Species Identification of Leishmania in Microscopy-Negative Smears from Suspected Cutaneous Leishmaniasis in Iran

Background: Direct microscopy remains a practical first-line test for cutaneous leishmaniasis (CL), but it can miss infections when parasite numbers are low or lesions are chronic. We evaluated whether PCR could detect and identify Leishmania in Giemsa-stained smears that had already been reported as microscopy-negative. Methods: We retrospectively analyzed 131 microscopy-negative smears from patients with clinically suspected CL who attended health-center laboratories in Gonbad-e-Qabus and Torbat-e-Jam, Iran, between March 2013 and February 2015. DNA was recovered from the stained slides by phenol- chloroform-isoamyl alcohol extraction. kDNA PCR was used for molecular detection and species identification. Results: Leishmania DNA was detected in 34 of 131 microscopy-negative samples (25.9%; exact 95% CI, 18.7%-34.3%). Twenty-three positive samples (67.6%) were identified as L. major and 11 (32.4%) as L. tropica. All L. major-positive samples came from Gonbad-e-Qabus, whereas all L. tropica-positive samples came from Torbat-e-Jam. Most PCR-positive lesions (27/34, 79.4%) were chronic, secondarily infected, and/or clinically atypical. In a small laboratory verification panel, all 10 microscopy-positive smears were PCR-positive and all 10 non-leishmanial dermatosis controls were PCR-negative. Conclusions: About one in four clinically suspected cases with a negative smear still had detectable Leishmania DNA. PCR of previously stained slides can therefore provide useful second-line confirmation and species information, especially when lesions are chronic or atypical. The small control panel supports assay performance in the study laboratory but is not large enough to provide precise estimates of clinical sensitivity or specificity.