Leishmania donovani and Cutaneous Leishmaniasis , Sri Lanka

I with Leishmania protozoa can result in cutaneous, mucocutaneous, or visceral leishmaniasis (VL), depending on the parasite, host, and environmental factors (1). Globally, the disease results in ≈2 million new cases and 2.4 million disability-adjusted life years each year (2). The leishmaniases have received renewed interest because of an upsurge of cases in traditionally leishmaniasisendemic areas and the emergence of new foci of disease (3,4). One of the most dramatic examples is a new focus of cutaneous leishmaniasis (CL) in Sri Lanka (5), from which >400 cases have been reported since 2001. Previously, multilocus enzyme electrophoresis (MLEE) characterization of a small number of isolates led to the surprising conclusion that CL in Sri Lanka was caused by Leishmania donovani (5). However, L. donovani typically causes VL, a potentially fatal disease and ongoing public health problem in neighboring India, Bangladesh, and Nepal, as well as in East Africa (1,2). No cases of VL have been reported in Sri Lanka. Occasional cases of CL due to L. donovani have been described in other VL-endemic regions (6–9). Karunaweera et al. (5) examined a limited number of isolates and used a single technique, MLEE. Although this technique is usually reliable for characterizing isolates, important exceptions were found in a recent study on L. donovani in East Africa (10). Therefore, we further investigated Sri Lanka CL by examining more isolates and using 2 molecular techniques. The Study Suspected clinical diagnoses of CL were confirmed by demonstrating the presence of Leishmania amastigotes in skin lesions, promastigotes in cultures, or both (5). Ethical approval was obtained from the Ethics Review Committee, Faculty of Medicine, University of Colombo. PCR, performed as described (11), confirmed 15 primary isolates as members of the genus Leishmania. Eight of the Sri Lanka isolates originated from Welioya (northeast), 1 from Jaffna (north), and 2 from Galle (south). DNA sequencing of a single-copy gene was used to identify the Leishmania species (10). The 6-phosphogluconate dehydrogenase (6PGDH) gene was chosen because it shows a high degree of sequence polymorphism among Leishmania species (12), is well represented in sequence databases, and is known to differentiate the main zymodeme from L. donovani in India (MON-2) from that elsewhere (13). Primers for conserved regions of 6PGDH were designed by using full-length gene sequences of the L. major FV1 (MHOM/IL/1980/Friedlin) and L. mexicana BEL21 (MHOM/BZ/1982/BEL21) reference strains. Primers 6PGDH-F (AAT CGA GCA GCT CAA GGA AG) and 6PGDH-R (GAG CTT GGC GAG AAT CTG AC) were designed to generate a 997-bp amplicon incorporating the 822-nt partial 6PGDH sequence that is represented for multiple Leishmania species in GenBank. The partial sequences of 6PGDH genes were obtained from 11 Sri Lanka isolates from patients with CL, 2 India isolates from patients with VL, and 2 additional known L. donovani strains. These 15 new sequences and 10 publicly available sequences for species belonging to the genus Leishmania were used to construct a classification (Figure 1). Of 17 L. donovani and L. infantum sequences, 14 were >99% identical and could not be separated; the remaining 3 stocks were from India and Bangladesh (Ind-1, Ind-2, and BG1) and clustered together with 58% bootstrap support. Thus, L. donovani from Sri Lanka formed a strongly supported group with L. donovani and L. infantum from Europe and Africa. This group was quite distinct from the group that includes L. major and L. tropica, which are the parasite species most closely related to L. donovani and L. infantum and which both cause CL in Africa and Asia. This analysis provided convincing evidence that all 11 Sri Lanka isolates examined were L. donovani or L. infantum. Strains of L. donovani from Sri Lanka were typed as zymodeme MON-37 by MLEE (5). This differs from the predominant India zymodeme (MON-2) in the mobility of 1 isoenzyme, 6PGDH. Therefore, the sequences were further analyzed to investigate the sequence variation underlying the isoenzyme identification. Translation of the 822-nt sequences showed 1 amino acid change that was consistent with the results of MLEE. A single nucleotide difference at position 976 was responsible for the occurDISPATCHES