Molecular identification of Leishmania major species in phlebotomine sand flies from Al Ahsa, Eastern KSA.

Objectives: Cutaneous leishmaniasis (CL) is considered an overlooked public health threat in KSA. CL is endemic to several Saudi regions, including Al Ahsa City, Eastern KSA. To our knowledge, no prior molecular identification of Leishmania parasites in vectors in Al Ahsa has been published. The aim of this study was to perform molecular identification of Leishmania species in sand flies collected from Al Ahsa. Methods: This cross-sectional study was conducted in Al Ahsa from July 2020 to May 2021. Sand flies were collected from the three areas with the highest rates of CL cases and sand flies, according to data from the Vector Borne Diseases Prevention Center in Al Ahsa. CDC miniature light traps and sticky traps were used to collect sand flies. Pools of 3-10 female sand flies were subjected to genomic DNA extraction. The extracted DNA was then amplified with a protocol targeting the Leishmania genus and using species-specific primers. For Leishmania species identification, a PCR-restriction fragment length polymorphism (PCR-RFLP) method was used. Results: Ten of 113 pools of samples tested positive for the Leishmania genus, according to our experimental protocol. Characterization of Leishmania species by PCR-RFLP established Leishmania major as the only species found in the collected sand flies. Conclusion: This is the first documentation of molecular identification of Leishmania species in phlebotomine sand flies in the Al Ahsa region. L. major was the only species identified in our study. Further comprehensive research investigating the vectors and reservoirs will be crucial to establish the dynamics of transmission of Leishmania in Al Ahsa.

Genotypic and phylogenic analyses of cutaneous leishmaniasis in Al Ahsa, Eastern Saudi Arabia during the coronavirus disease 2019 pandemic: First cases of Leishmania tropica with the predominance of Leishmania major.

During the coronavirus disease 2019 lockdown period, a surge in sandflies and cutaneous leishmaniasis (CL) cases was observed in Al-Ahsa, Saudi Arabia. Skin punch biopsies were obtained from 100 patients clinically diagnosed with CL in Al-Ahsa who had no travel history in the last 6 months. Impression smears were used following a three-step polymerase chain reaction (PCR) protocol using genus-specific primers targeting kDNA and ITS1. Leishmania speciation was determined by ITS1 PCR/nested PCR-restriction fragment length polymorphism and sequencing. A phylogenetic tree was constructed. The associated patient characteristics were analyzed. Using internal transcribed spacer one (ITS1)-PCR/nested PCR, 98 cases were considered true-positive CL. Leishmania major was the predominant species, and Leishmania tropica was identified in three cases. Microscopy had poor sensitivity and perfect specificity. Direct ITS1-PCR missed nine cases. Sex, residence, and treatment outcome were significantly associated with the occurrence of Leishmania; distribution of skin lesion(s) and treatment outcome were significantly associated with Leishmania genotype. This is the first time that L. tropica was identified as a cause of CL in human in Al-Ahsa, in addition to the predominant zoonotic species, L. major. We recommend using ITS1-nested PCR for negative cases by ITS1-PCR. Further exploration of Leishmania transmission dynamics in vectors and reservoir animals is essential for designing effective preventive measures.