Molecular Diagnosis of Cutaneous Leishmaniasis and Species Identification: Analysis of 54 Histology Negative Skin Biopsies.
Lamis Yahya, Mohammad Hourieh, Wasim Raslan, Marwan El-Sabban, Ibrahim Khalifeh. American University of Beirut, Lebanon; Tishreen University, Lattakia, Syrian Arab Republic; Aramco, Dhahran, Saudi Arabia
Background: Cutaneous Leishmaniasis (CL) is endemic in the Middle East and North Africa and displays a wide spectrum of clinical manifestations. Confirming the diagnosis of CL histologically depends on the identification of the amastigotes, which may be inconclusive. The number of amastigotes may vary significantly depending on the strain type, host response & the disease stage. Accurate histological diagnosis is significant due to the requirement of targeted treatment.
Design: Skin biopsies from 122 patients from Lebanon, Syria, and Saudi Arabia with suspected untreated CL were reviewed. Clinical data includes age, gender, duration of the lesion, and biopsy type (shaved biopsy, SB versus punch biopsy, PB). Seven sections from each formalin fixed paraffin embedded skin biopsy (FFPE) were stained: 3 H&E, 1 Giemza, 1 AFB, 1 GMS & 1 PAS. All cases were reviewed by 2 pathologists and classified according to the modified Ridley's parasitic index (scale 0 to 6). DNA was extracted using a standard protocol from ribbons originating from the respective FFPE. PCR was performed using primers specific for the Leishmania ribosomal internal transcribed spacer 1 (ITS1-PCR). The digestion of the ITS1-PCR amplicons with the restriction enzyme HaeIII was performed for restriction fragment length polymorphism (RFLP) analysis and subsequent sub-speciation.
Results: Out of 122 skin biopsies, 54 cases (44.3%) showed a parasitic index of 0 to 1+ (no unequivocal amastigotes detected). Of the negative cases, 9 were SB and 45 were PB. The age ranged from 2 to 90 years (mean = 35 years, SD= 24.0 & males/females ratio is 6/5). The duration of the lesion ranged from 1 month to 60 months (mean= 10 months, SD= 14.6). ITS1 PCR was positive for all 54 cases (100% sensitivity). RFLP analysis identified Leishmania tropica sub-species in all cases.
Conclusions: Patients with clinically suspected CL, whose skin biopsies failed to detect Leishmania amastigotes, is a commonly encountered problem that represented 44.3% of the cases included in our study. The histologic confirmation of CL is crucial especially with the wide clinical manifestations and the availability of targeted therapy. In this study, we describe all stages of an optimized protocol from DNA extraction to sub-speciation by RFLP. According to our results, ITS1 PCR usage showed high sensitivity and specificity in confirming the diagnosis of CL where histology failed to detect Leishmania amastigotes.
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 130, Wednesday Afternoon