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

Abstract

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.

Figure 1

PCR-based Protocol for identification and speciation of Leishmania (Modified from Schönian et al., 2003 and El-Beshbishy et al., 2013)^,.

Figure 2

Geimsa stained impression skin smears showing Leishmania species amastigotes (Circled).

Figure 3

MetaPhor (4%) Agarose gel for Leishmania speciation isolated from clinical specimen of CL patients by analysis of restriction pattern of ITS1 PCR/nPCR products using restriction enzyme (HaeIII). Lane 1: 50 bp DNA ladder; lane 2: L. major MHOM/TM/82/ Lev positive control (203 and 132 bp); lane 3: L. tropica MHOM/SU/80/K28 positive control (200 and 57 bp); lanes 4–6 & 8–12: L. major samples; lane 7: L. tropica sample.

Figure 4

Dendrogram shows the Neighbor-joining phylogenetic tree of L.major (SA-L.m 1–9 and 39–98 in one cluster, 10–38 in one cluster, 99, 100 and 101) and L.tropica (SA-L.t 44, 77 and 78) study isolates relied on ITS1-5.8S rDNA gene sequences in comparison to reference strains (their accession number is located before their names). Similar sequences were clustered in the tree. Bootstrap analysis was relied on 1000 replicates. The distance scale at the bottom of the tree represents the number of differences between the sequences.

Figure 5

Different skin manifestations of CL patients. A: Right leg nodular lesion (1.5–2 cm) in a 43-year-old male laborer; B: Right leg ulcerative plaque lesion (4 cm) in a 37-year-old male farmer; C: Left forearm plaque lesion (0.5–1 cm) in a 48-year-old male truck driver; D: Left leg ulcerative lesion (5–5.5 cm) in a 31-year-old male driver.

Figure 6

Resolution of the CL lesions. A-C: Post inflammatory hyperpigmentation and scaring in a different exposed body sites after a full course of treatment. (Once weekly 0.5 mL per lesion (50 mg) intralesional sodium stibogluconate for 6 weeks course).