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 southern 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, and L. donovani parasite species have been shown to cause Old 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 rehydrated in 200 μL 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 μg/mL) while on the slide. The DNA was then precipitated with isopropyl alcohol, centrifuged, and resuspended 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, and L. 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 fragment. 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, β-actin was amplified from each sample.13
In addition to identifying Leishmania species on the basis of PCR product size, representative PCR products were sequenced for species confirmation. Because of the difficulties with direct sequencing of the PCR products using the Uni21 and Lmj4 primers, we cloned the gel-purified PCR reaction into pCR4-TOPO vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. The clones were sequenced directly using M13 forward and reverse primer pair. The resulting sequence was analyzed using DNASTAR sequence analysis software (DNASTAR, Inc., Madison, WI). Sequences were compared with published sequences from kinetoplast DNA (kDNA) from L. major (GenBank accession no. J04654), L. infantum (GenBank accession no. AF188701), L. tropica (GenBank accession no. Z32841), and L. donovani (GenBank accession no. AF167718).
Of 85 archived Giemsa-stained, microscopically confirmed slides, 41 (48%) were found to contain Leishmania DNA by PCR. All 41 PCR products were approximately 650 bp in size, comparable with the L. major DNA control and clearly different in size than the L. donovani DNA control and expected sizes for the other Leishmania species (specifically L. infantum and L. tropica, which are indistinguishable from L. donovani using this primer set), indicating that these patients were infected with L. major (Figure 1). To confirm the presence of L. major DNA, four randomly selected samples were sequenced. Based on PCR product size and sequence confirmation, these findings suggest that L. major is the predominant, if not the exclusive, species responsible for CL in Mali.
PCR-confirmed samples came from various geographic and ecoclimatic zones across the country (north Sudan savanna, Sahelian areas, and sub-Saharan zones) (Figure 2), with the exception of the southern part of the country (south Sudan savanna). The majority came from the Sahelian areas and North Savanna areas. Figure 2 does not suggest that CL is found only in the locations indicated, because regions farther away from the Koulikorou region (the location of CNAM) may not be adequately represented because of the self-reporting nature of CL in Mali.
Species identification is important for characterizing the epidemiological and clinical spectrum of CL in Mali. As confirmed by our study of archived samples, CL is endemic in several regions of Mali; however, the disease is underreported and unknown to medical personnel in remote areas. Typically, only patients with chronic and persistent CL are referred to the CNAM dermatology clinic after treatment failure or as a result of misdiagnosis. Thus, CL cases received from this clinic provide a good representative sample of the geographic distribution of chronic CL in Mali. Species identified in these patients may also be a good representative sample of the parasite population responsible for the disease.
We used PCR to test for the presence of four species (L. major, L. tropica, L. infantum, and L. donovani) potentially responsible for CL in Mali. Of the 85 slides identified as positive by microscopy, we confirmed the presence of Leishmania DNA in 41 archived samples. The presence and integrity of DNA in the Leishmania PCR-negative samples were established by PCR using a housekeeping gene, β-actin (data not shown). At the onset of this project, DNA was extracted from only one of three skin scrapings on each Giemsa-stained slide. Despite the possibility that not all specimens on each slide contained amastigotes, we decided to pursue this study design, because our objective was not to confirm clinically and microscopically suspected cases but identify the diversity of Leishmania species; PCR confirmation of over 40 specimens was sufficient for this purpose. In addition to the study design, it is possible that PCR, at least in the context of our protocol, is not as sensitive as Giemsa for the detection of Leishmania DNA. Whereas the presence of a single amastigote is sufficient to deem a specimen positive by microscopy, it is possible that many more parasites are needed for detection with our PCR method. This finding may be especially true in cases of chronic CL, where the parasite burden per lesion is much lower. The primer pair used in this study has been shown to reliably detect numerous Leishmania species.12 We tested the primer pair on L. major and L. donovani DNA and showed that we were able to amplify DNA fragments from these two Leishmania species. Therefore, it is unlikely that our primer pair selectively detected L. major in our microscopy-confirmed cases.
Using the kDNA PCR, one of the most sensitive PCR methods to detect Leishmania DNA,12,14 we found that L. major is the only species present in archived specimens from individuals diagnosed with CL at CNAM. The dermatology clinic of CNAM is the only national institution specializing in the treatment of CL and other skin diseases in Mali. Access to CNAM is difficult for much of the population in Mali, specifically from the northern regions. Thus, other Leishmania species may exist in Mali, but they seem to be rarely observed in the geographic areas where L. major was found. Along with three previous reports confirming the presence of L. major in Mali,7–9 this study suggests that L. major is likely the sole species responsible for CL in Mali and that it is found throughout the country.
We would like to thank Bourama Traore, Ibrahim Sissoko, and Cheick Coulibaly from the Leishmaniasis Laboratory, International Center of Excellence in Research (ICER-Mali) for their technical assistance with this study. We also thank Drs. Shaden Kamhawi and Fabiano Oliveira for technical assistance and reviewing this manuscript. This research was supported in part by the Intramural Research Program of the National Institutes of Health and the University of Bamako.
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