Short Report: Leishmania major, the Predominant Leishmania Species Responsible for Cutaneous Leishmaniasis in Mali

Leishmania major is the only species of Leishmania known to cause cutaneous leishmanisis (CL) in Mali. We amplified Leishmania DNA stored on archived Giemsa-stained dermal scraping slides obtained from self-referral patients with clinically suspected CL seen in the Center National d'Appui A La Lutte Contre La Maladie (CNAM) in Bamako, Mali, to determine if any other Leishmania species were responsible for CL in Mali and evaluate its geographic distribution. Polymerase chain reaction (PCR) amplification was performed using a Leishmania species-specific primer pair that can amplify DNA from L. major, L. tropica, L. infantum, and L. donovani parasites, possible causative agents of CL in Mali. L. major was the only species detected in 41 microscopically confirmed cases of CL from five regions of Mali (Kayes, Koulikoro, Segou, Mopti, and Tombouctou). These results implicate L. major as the predominant, possibly exclusive species responsible for CL in Mali. Cutaneous leishmaniasis (CL) is a skin infection caused by the hemoflagellate Leishmania, which is transmitted by the sand fly bite. CL lesions are characterized by an ulcer with indurated borders and a necrotic base that often heals without treatment months after the initial infection. The geographic distribution of CL is quite varied and found in 88 countries on five continents, with 1.5-2.0 million new cases reported yearly worldwide. CL is often divided into Old World (including south- ern Europe, the Middle East, parts of southwest Asia, and Africa) and New World (from southern United States through Latin America to South America) forms depending on the geographic setting of the infection. Approximately 20 different Leishmania species cause CL, although the species responsible for New World and Old World CL are distinct. For example, L. tropica, L. major, L. aethiopica, L. infantum ,a ndL. donovani parasitespecies havebeen showntocauseOld World CL. 1 Epidemiological studies of CL using the leishmanin skin test have shown that CL is widely distributed throughout Mali, particularly in the northern Sahelian areas. 2-6 L. major is the only Old World Leishmania species detected in Mali. Before 2009, L. major had been isolated from two skin lesions, one from a visitor to Mali and the other from a Malian. 7,8 A more recent study identified four different L. major strains in Mali. 9 Although these reports established the presence of L. major in the country, the frequency of specific etiologic agents and the extent of their geographical distribution in Mali remain unknown. In this study, we used Leishmania species-specific polymerase chain reaction (PCR) to confirm the presence and distribution of L. major and determine if other Leishmania species exist or are clinically relevant in Mali. We show that L. major is the predominant and probably exclusive species of Leishmania found in Mali. Moreover, L. major was found in various geographic and ecoclimatic zones across the country. Eighty-five archived anonymized Giemsa-stained skin scrapings of individuals with microscopically confirmed CL who presented to the Center National d'Appui A La Lutte Contre La Maladie (CNAM) in Bamako, Mali between January of 2005 and March of 2006 were used in this study (National Institutes of Health Institutional Review Board exemption #11605). As part of the National Ministry of Health, CNAM is the only referral clinic for skin diseases in Mali and receives patients from the entire country. Parasite DNA extraction was performed using the techniques described elsewhere. 10,11 Briefly, Giemsa-stained specimens were rehy- drated in 200 mL lysis buffer (100 mM Tris HCl (pH 7.5), 5 mM ethylenediaminetetraacetic acid (pH 8.0), 0.2% sodium dodecyl sulfate, 200 mM NaCl, and Proteinase K freshly added at 200 mg/mL) while on the slide. The DNA was then precipitated with isopropyl alcohol, centrifuged, and resus- pended in water. Leishmania DNA was amplified by PCR as described by Anders and others. 12 The Uni21 and Lmj4 primer pair amplifies L. major, L. tropica, L. infantum ,a ndL. donovani DNA. 12 Amplified DNA from each Leishmania species differs in size, allowing for species identification based on the length of the PCR fragment. 12 For example, L. major DNA yields a 650-bp product, whereas L. donovani DNA produces an 800-bp frag- ment. The positive controls were DNA from cultured L. major and L. donovani. The negative control was a blank sample (water) processed in parallel with our test samples from the beginning of the DNA extraction. To control for sample DNA quality,b-actin was amplified from each sample. 13