A 4-year-old girl presented with a 2 month history of increasing swelling and painful ulcer on the right upper lip. On clinical examination, an erythematous infiltrated, crusted, scaled and fissured plaque of the upper lip leading to macrocheilitis was observed. Cervical lymph nodes were noticed to be enlarged (Fig. ). With suspicion of granulomatous macrocheilitis; slit impression smear, culture and PCR were achieved. Just PCR was positive and characterized the parasite as L. infantum.\nThe studied patients were examined in the Dermatology Clinic of Saadi Hospital and Fajr Health Center from January 2014 to the end of December 2015. All patients were came from endemic regions of Fars province. A structured questionnaire was used to collect clinical and therapeutic data from all patients.\nThe margin dermal scrapings were prepared with a no. 15 disposable sterile surgical blade (Unicut, Chicago, USA) to make a slit in the border of the lesion. Dermal tissues were stained with Giemsa (Merck, Darmstadt, Germany), and exhibited amastigotes by light microscope. Employing Giemsa, amastigotes are seen within the cytoplasm of macrophages as pale blue oval bodies with a dark blue nucleus and a small rod-shaped kinetoplast with a specified mitochondrial frame that contains extra-nuclear DNA.\nIn addition, an aspirate from the active indurated margins of the same lesions was transferred to two tubes of the modified NNN culture medium. Modified NNN medium consists of two phases, horse blood agar base and an overlay Locke’s solution. The specimens were inoculated into the liquid phase of the biphasic medium and incubated. Positive cultures were run in RPMI-1640 medium (Gibco, Frankfurt, Germany) supplemented with 10% heat inactivated FCS (Gibco, Frankfurt, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Frankfurt, Germany) for mass cultivation. Promastigotes were harvested and kept at -20 °C until used.\nTotal genomic DNA was extracted from each clinical sample using the AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea), according to manufacturer’s instructions. The quantification and quality control of the DNA extraction procedures were performed using a nano spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The DNA was stored at −20 °C until being used.\nThe conserved area of the minicircle kDNA from the Leishmania species was amplified by Semi-Nested PCR. The primers LINR4 (forward) (5’-GGG GTT GGT GTA AAA TAG GG-3’), LIN17 (reverse) (5’-TTT GAA CGG GAT TTC TG-3’), and LIN19 (reverse) (5’-CAG AAC GCC CCT ACC CG-3’) were used for amplification []. Standard PCR was carried out with 40 cycles, each consisting of 30 s at 94 °C, 30 s at 52 °C (for LINR4 and LIN17) or 58 °C (for LINR4 and LIN19), 1 min at 72 °C, and a final extension at 72 °C for 10 min in an Eppendorf thermal cycler (Hamburg, Germany). PCR products were visualized by UV after electrophoresis on 1.5% agarose gel using TAE buffer and staining with GelRed (Biotium Inc., Hayward, CA). All primers were synthesized by Macrogen Genomics Laboratories (Macrogen, Seoul, Korea). A 650-bp fragment was amplified for L. major, whereas a 760-bp and 720-bp fragments were amplified for L. tropica and L. infantum, respectively (Fig. ).\nThe PCR products were purified by using QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) and sequenced through the sequencing service of Macrogen Genomics Laboratories (Seoul, Korea). The resulting sequences were aligned and compared with those of existing sequences related to Leishmania in GenBank. A Maximum Likelihood (ML) tree was constructed using the MEGA-7 program [], and genetic distances were calculated with the Maximum Composite Likelihood method. The sequences of three patients of L. tropica showed 100% identity to the published isolate IranJWtrop (AB678350). Also, these three isolates showed 99% identity to the published isolates from UK, Egypt, and Iran (AF308689, X84845, and KM491168) (Fig. ). The sequences of three patients of L. major showed 100% identity to the published isolate IranJWmaj (AB678349), In addition, the three L. major showed 99%, 99% and 98% identity to the published isolates from UK and Iran (AF308685, KM555292, and KM555295) respectively (Fig. ). The sequence of L. infantum showed 99% identity to the published isolate IranJWinf (AB678348). Plus, this isolate showed 98% identity to the published isolate from Spain (EU437407) (Fig. ).\nThe patients received intramuscular injections of Meglumine antimoniate (Glucantime®; Sanofi-Aventis, Paris, France), 20 mg/kg/day over a 3-week period with good results [, ]. All lesions resolved after 3 weeks of therapy. After 1 year of follow up, the patients were completely normal and no relapses were observed.
A 28-year-old man expressed displeasure about a swelling localized to the left side of the upper lip. The patient also stated that the swelling had appeared about 2 months before and that it rapidly enlarged up to the current morphology and size. Clinical examination showed the presence of a swelling which involved the left side of the upper lip. The surface of the swelling was slightly erythematous and irregular, because of the presence of lesion and scaling (Fig. ). Consistency was parenchymatous-hard. With suspicion of mycotic infection, laboratory tests were carried out. Slit dermal smears and culture were positive. PCR characterized the parasite as L. tropica.