Molecular Detection of Cutaneous and Mucocutaneous Leishmaniasis in an Endemic Areas of Pakistan

Background: Leishmaniasis is a neglected tropical disease with a substantial burden in endemic regions such as Pakistan. Accurate diagnosis and species identification are essential for appropriate case management, epidemiological surveillance, and disease control. Objective: To determine the prevalence and species distribution of cutaneous and mucocutaneous leishmaniasis in endemic regions of Pakistan using SYBR Green real-time polymerase chain reaction (RT-PCR). Methods: This cross-sectional laboratory-based study included 171 clinically suspected patients presenting with cutaneous or mucocutaneous lesions between 2018 and 2019. Clinical specimens were examined by Giemsa-stained microscopy, culture, and SYBR Green RT-PCR targeting the internal transcribed spacer 1 (ITS1) region for species identification. Demographic, clinical, and geographic data were recorded, and descriptive statistics were used to summarize prevalence and distribution patterns. Results: Of the 171 suspected cases, 100 (58.4%) were positive by RT-PCR, compared with 59 (34.5%) by microscopy and 40 (23.3%) by culture. Cutaneous leishmaniasis accounted for 136 cases (79.5%), mucocutaneous leishmaniasis for 31 (18.1%), and mixed presentations for 4 (2.3%). Dry lesions predominated, occurring in 136 patients (79.5%). The highest frequency of cases was observed in the 20-40-year age group (26.9%). Species identification among PCR-positive cases showed Leishmania tropica in 64%, L. major in 22%, L. infantum in 11%, and unclassified species in 3%. The greatest geographic burden was observed in Khyber Pakhtunkhwa. Conclusion: SYBR Green RT-PCR is a sensitive and reliable method for the detection and species-level identification of leishmaniasis. The predominance of L. tropica and the marked concentration of cases in Khyber Pakhtunkhwa emphasize the need for strengthened molecular diagnostics, surveillance, and targeted control strategies in endemic settings.