Diagnostic Performance of Microscopy, Culture, Histopathology, and PCR in Cutaneous Leishmaniasis: Evidence from an Endemic Region in Libya

Cutaneous leishmaniasis is a neglected tropical disease with a significant public health impact in many endemic regions. Its diagnosis traditionally relies on clinical evaluation supported by microscopic examination of amastigotes obtained from the edges of skin lesions. Although this approach is simple, inexpensive, and quick, it has low sensitivity. Other conventional methods, such as culture and histopathology, provide additional diagnostic information but also have limitations in sensitivity, specificity, and practicality. In recent years, molecular techniques, especially polymerase chain reaction (PCR), have changed the diagnostic landscape of infectious diseases, including cutaneous leishmaniasis. PCR offers high sensitivity and specificity and can identify Leishmania species. However, its use is limited by high costs, longer processing times, and the need for specialized laboratory facilities and trained personnel. Given these challenges, this study aimed to evaluate and compare the diagnostic performance and practical applicability of four diagnostic methods for cutaneous leishmaniasis: slit-skin smear microscopy, culture, histopathological examination, and polymerase chain reaction (PCR). PCR was used as the reference standard to assess the diagnostic performance of slit-skin smear microscopy, culture, and histopathology by determining their sensitivity and specificity. A comparative diagnostic study was conducted between June 2024 and January 2025 at Tripoli Central Hospital and the National Center for Disease Control (NCDC), Libya. Patients of all ages and both genders with clinically suspected cutaneous leishmaniasis and no prior treatment were enrolled. Clinical and demographic data were collected, and lesion samples were examined using slit-skin smear microscopy, histopathology, parasite culture, and polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). The sensitivity of each diagnostic method was calculated using PCR-RFLP as the reference standard. Among 42 patients, PCR-RFLP demonstrated the highest sensitivity, detecting Leishmania in (41 sample) 97.6% of cases; (L. major, 92.7 %; L. tropica, 7.3%), followed by slit-skin smear (88%), histopathology (76.1%), and culture (42.8%). Females (59%) were more affected than males (41%), and children aged 0–9 years were the largest group. Most cases originated from Kikla and Gerian, with the majority of patients lasting about 8 weeks before seeking medical advice. The face and extremities were the most commonly affected sites, with ulcerated nodules being the predominant clinical presentation type, and PCR proved to be the most reliable diagnostic tool. PCR demonstrated superior sensitivity and reliability compared to conventional methods, underscoring its value as the most effective diagnostic tool for cutaneous leishmaniasis, particularly for species identification and confirmation in endemic regions.