Comparative evaluation of parasitological, serological, and molecular methods for the diagnosis of cutaneous leishmaniasis in the Garmian area, Kurdistan region of Iraq.

Background: Cutaneous leishmaniasis (CL) is a neglected tropical disease and remains a significant public health issue in many endemic regions. Accurate diagnosis is often challenging due to the broad spectrum of clinical manifestations, which can resemble other dermatological conditions. Therefore, highly sensitive and specific diagnostic methods are essential to ensure proper identification and management of the disease. Methods: This cross-sectional study was conducted between December 2019 and March 2020 at Kalar General Hospital in the Garmian area, Kurdistan Region of Iraq, and enrolled 85 patients with clinically suspected CL. Diagnosis was based on dermatological examination or ongoing antileishmanial treatment. Specimens were collected from cutaneous lesions using fine needle aspiration for direct microscopic examination and molecular analysis. Additionally, venous blood samples were obtained for serological testing using enzyme-linked immunosorbent assay (ELISA). Diagnostic performance was assessed for four methods: direct microscopy, ELISA, conventional polymerase chain reaction (PCR), and nested PCR. Nested PCR served as the comparative reference method rather than an independent gold standard; agreement of the other assays with nested PCR was expressed as positive percent agreement (PPA) with 95% confidence intervals and Cohen's κ. Results: Among the 85 patients, CL was confirmed in 84 (98.82%) cases by nested PCR, 71 (83.53%) by conventional PCR, 64 (75.29%) by direct microscopy, and 45 (52.94%) by ELISA. Relative to nested PCR, the positive percent agreement was 84.5% for conventional PCR, 76.2% for direct microscopy, and 53.6% for ELISA. Because only one case was nested-PCR-negative, specificity could not be reliably estimated and is not reported. Conclusions: Nested PCR showed the highest detection rate and is a valuable tool for CL diagnosis, particularly in cases with low parasite load. Because nested PCR served as the reference method, formal diagnostic accuracy could not be established, and larger studies employing an independent reference standard (culture, sequencing, or a composite standard) are warranted. In contrast, ELISA showed poor diagnostic performance and should not be relied upon as a standalone test for CL. Incorporating molecular techniques, especially nested PCR, into routine diagnostic protocols is recommended to improve early detection and effective management of cutaneous leishmaniasis.