Molecular diagnosis of cutaneous leishmaniasis and species identification: analysis of 122 biopsies with varied parasite index

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Abstract
Background: Cutaneous leishmaniasis is endemic in the Middle East and North Africa. Confirming the diagnosis histologically depends on amastigote identification, which varies significantly depending on the inoculum, strain type, host response and disease stage. Accurate histological diagnosis is mandatory for appropriate therapy. Methods: Skin biopsies from 122 patients from Lebanon, Syria and Saudi Arabia with clinical diagnosis of untreated leishmaniasis were reviewed and clinical data extracted. Cases were classified according to the modified Ridley's parasitic index. DNA was extracted from formalin‐fixed paraffin‐embedded blocks. Polymerase chain reaction (PCR) was performed using Leishmania‐specific ribosomal internal transcribed spacer 1 (ITS1‐PCR). Nested ITS1‐PCR was performed on cases negative for conventional ITS1‐PCR. ITS1‐PCR amplicons were digested with HaeIII for subsequent restriction fragment length polymorphism (RFLP) subspeciation. Results: Of 122 cases, 54 (44.3%) showed a parasitic index of 0–1+ (no unequivocal amastigotes). ITS1‐PCR (conventional and nested) was positive for all cases as compared with negative control tissue. RFLP identified Leishmania tropica in all cases. Patients with clinically suspected leishmaniasis, whose skin biopsies failed to detect amastigotes represented 44.3% of our cases. Conclusions: In this study, we describe a rapid and optimized protocol from DNA extraction to leishmaniasis subspeciation. ITS1‐PCR showed high sensitivity and specificity in confirming clinically suspected cases. Yehia L, Adib‐Houreih M, Raslan WF, Kibbi A‐G, Loya A, Firooz A, Satti M, El‐Sabban M, Khalifeh I. Molecular diagnosis of cutaneous leishmaniasis and species identification: analysis of 122 biopsies with varied parasite index.