Molecular Detection of Leishmania Species on Microscopically Negative Smears in Gonbad-e-Kavus, Northeastern Iran

Background: The diagnosis of cutaneous leishmaniasis (CL) is typically based on microscopic examination of Giemsa-stained smear. However, this method can lead to false negative and false positive results. Therefore, the study aimed to identify L. major and L . tropica species by investigating microscopically negative smears suspected of CL using the multiplex nested polymerase chain reaction (PCR) method. Materials and Methods: This study was conducted on 227 microscopically negative specimens from individuals suspected of having CL referred to the CL Treatment and Diagnosis Center in Gonbad-e Kavus Health Center, Golestan Province, northeastern Iran. Multiplex nested PCR technique was utilized to detect L. tropica and L. major under investigation based on ITS1 sequence. Results: 2.64% (6/227) of specimens were detected as positive for Leishmania species, all of which were identified as L . major . Conclusion: The multiplex nested PCR technique demonstrated that microscopic examination of the smears is linked to false negative in diagnosing CL. Consequently, it is recommended to utilize the multiplex nested PCR method as either a primary diagnostic or a confirmatory approach for cases that are microscopically negative.