Cutaneous leishmaniasis caused by Leishmania (L.) major infection in Sindh province, Pakistan

Leishmaniasis is endemic in Pakistan and is wide-spread throughout the country. Polymerase chain reaction (PCR) was performed to identify the Leishmania species present in cutaneous leishmaniasis (CL) patients from new endemic areas of the central part of Sindh province, Pakistan. The PCR primers used were designed for the identification and differentiation of Leishmania (Leishmania) major and Leishmania (Leishmania) tropica species, and PCR bands at 620 and 830 bp of the parasite-specific kinetoplast DNA sequences was identified for L. (L.) major and L. (L.) tropica, respectively. Among a total of 144 DNA samples purified from the skin biopsies of clinically suspected CL patients, 108 (75%) were positive for PCR amplification. Out of the 108 cases, 105 (97.2%) were determined to be positive for L. (L.) major infection, and 3 (2.8%) were positive for L. (L.) tropica infection. It was concluded that CL caused by L. (L.) major is the main source of infection in the central part of Sindh province in Pakistan. This rapid screening technique could be used for the diagnosis of a large number of samples from skin lesions, which commonly contain other bacterial and fungal infections.     

Amplified fragment length polymorphism (AFLP) analysis is useful for distinguishing Leishmania species of visceral and cutaneous forms

The Leishmania strains belonging to cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been reported to possess close homology in genome profiles. To confirm this on genetic basis an attempt was made to differentiate Leishmania major; Leishmania tropica and Leishmania donovani genetically for the first time using amplified fragment length polymorphism (AFLP)—a high throughput DNA fingerprinting technique. The objective of this research work was to identify DNA markers of CL and VL. Ten combinations of selective primers detect a total of 1487 informative AFLP marker. Percentage of polymorphism was 45.12%. Three hundred and thirty-seven unique AFLP markers were also identified in three species of Leishmania. A clear distinction was revealed between L. major and L. donovani. It was inferred by AFLP analysis that a higher rate of polymorphisms occurred among Leishmania species which indicate the distinguished pattern of the disease cause by Leishmania, i.e. VL and CL. Analysis based on polymorphic AFLP markers revealed considerably high genetic variation among the genome of these species which was sufficient to distinguish between CL and VL.     

A molecular and parasitological survey on cutaneous leishmaniasis patients from historical city of Kashan in Isfahan province,center of Iran

ObjectiveTo study cutaneous leishmaniasis (CL) for identifying the dominant Leishmania species on CL patients referred to medical health centers of historical Kashan city and suburbs located in Isfahan province in central part of Iran during 2010 to 2011.MethodsFrom 137 CL cases, were microscopically positive, the skin lesion serosity materials of 103 cases were cultured in monophasic culture media (RPMI 1 640). We used the PCR-RFLP method for characterization the Leishmania isolates, by using specific internal transcribed spacer (ITS1) primers and HAEШ as the restriction fast enzyme. DNA was extracted from 63 samples.ResultsL. tropica is main species in 58 (92.1%) cases and L. major is identified in 5 (7.9%) cases. Indeed randomly two isolates were the species characterized as L. major produced ulcer at the base tail of BALB/c mice after 3 weeks but from three L. tropica isolates none of them produced any lesion during 6 months post inoculation.ConclusionsThe parasitological, epidemiological aspect and molecular methods of this study showed that, Kashan and suburb are anthroponetic CL area despite this city located in Isfahan province as an ancient focus of zoonotic CL in Iran.     

Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran

Background

Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis.

Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran

Objective To determine the geographical distribution of Leishmania species causing cutaneous leishmaniasis (CL) and to study the genetic heterogeneity of Leishmania major isolates from different endemic areas of Iran. Methods A total of 341 isolates from lesions of patients living in 11 provinces of Iran were grown in culture medium and inoculated to BALB/c mice to detect possible visceralisation. The species were identified by isoenzyme analysis using a battery of six enzymes and kinetoplast (k) DNA‐PCR technique. Genetic variation among L. major isolates was analysed by random amplified polymorphic DNA (RAPD) technique. Results Of the total 341 isolates, 283 isolates were L. major and 58 isolates were Leishmania tropica. In rural areas, the causative agent of CL was mainly L. major (95%L. major vs. 5%L. tropica), in urban areas it was L. tropica (65%L. tropica vs. 35%L. major). All isolates of L. major and 8.6% of L. tropica isolates showed visceralisation in BALB/c mice. There is considerable genetic diversity between L. major strains from different endemic areas and even between some isolates of the same endemic area. Conclusion Leishmania major is the most frequent species in the endemic areas of CL in eleven provinces of Iran, and genetic diversity is a common feature of L. major in the country.     

Identification of Old World Leishmania species by PCR–RFLP of the 7 spliced leader RNA gene and reverse dot blot assay

The aim of the study was to assess the 7SL RNA PCR followed by restriction fragment length polymorphism (RFLP) and reverse dot blot (RDB) assays for use in identification of Old World Leishmania species. Species‐specific RFLP patterns were obtained for Leishmania major, Leishmania tropica and the Leishmania donovani complex when the 7SL RNA PCR product was digested with the restriction enzyme BsuRI, an isoschizomer of HaeIII. For the RDB assay, biotin‐labelled 7SL RNA amplicons were hybridized to Leishmania genus‐specific and species‐specific oligonucleotide probes immobilized onto a membrane. The Old World Leishmania species could be distinguished by using five probes: one that was a genus‐specific probe and hybridized to all Leishmania species (Lc), two that were specific for L. major (Lm1 and Lm2), one that was specific for L. tropica (Lt) and one that detected both L. major and L. tropica (Lmt). The PCR–RDB was 10 times more sensitive than 7SL PCR and can detect <1 parasite. In addition, the identification of species was easier and more reliable than with 7SL PCR–RFLP. 7SL PCR–RFLP detected parasites in 50 of 57 clinical samples, whereas PCR–RDB detected 53 and 55 were detected by amplification of kinetoplast (k) DNA. The 7SL RNA PCR has proven useful for direct diagnosis of Old World leishmaniasis, especially when combined with the RBD assay for species identification.     

Comparison of Clinical Samples and Methods in Chronic Cutaneous Leishmaniasis

This study aimed at finding out the most effective clinical samples and methods in chronic cutaneous leishmaniasis (CCL). Smear, aspiration fluid, and filter paper samples were taken from 104 skin lesions of suspected cases with CCL, and they were compared using microscopic examination, culture, and molecular methods. We characterized four different forms of CCL and identified the causative agents in CCL forms using high-resolution melting curve real-time polymerase chain reaction assay. We observed that smear was detected to be the most sensitive (63.5%) among clinical samples, and real-time polymerase chain reaction method was the most sensitive (96.8%) among the methods used in diagnosis of CCL. We identified 68.8% Leishmania tropica and 31.2% L. infantum in papular lesions, 69.2% L. infantum and 30.8% L. tropica in nodular lesions, 57.9% L. tropica and 42.1% L. major in ulcerating plaque lesions, and 55.5% L. tropica and 44.5% L. major in noduloulcerative lesions in CCL patients.     

Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis

Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research.     

An overview of a diagnostic and epidemiologic reappraisal of cutaneous leishmaniasis in Iran

Cutaneous leishmaniasis (CL) is a widespread tropical infection which has a high incidence rate in Iran. Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (ACL), and Leishmania major, which causes zoonotic cutaneous leishmaniasis (ZCL), are endemic in various parts of Iran with a high incidence rate. The aim of this study was to evaluate the reappraisal of the diagnosis and epidemiology of CL in Iran, by different clinical, parasitological and molecular assays among patients suspected of CL referred to the Department of Parasitology, at the Pasteur Institute of Iran during 2006–2009. Two hundred samples from patients with ulcerative skin lesions were collected, clinical analyses were applied, data questionnaire was completed and samples were examined for CL by using both direct microscopic and culture methods. Moreover, PCR assay was applied for detection of Leishmania species in CL isolates resulting from parasitological assay. Clinical observation revealed that the majority (58%) of lesions was single; double lesions were observed in 22% of patients, and only 20% of CL had multiple lesions. Out of 200 patients, Leishman body was observed in 77 samples (38.5%) by direct smear and 40% by cultivation assay. Most patients (21.3%) had a travel history to the Isfahan province, one of the most important endemic areas of CL located in center of Iran. PCR assay by kDNA indicated 32 and 18 out of 50 isolates respectively had similar patterns with standard L. major and L. tropica. In conclusion, clinical manifestations and an appropriate diagnostic assay with a parallel molecular characterization of CL may lead to a screening evaluation of disease, prognosis, treatment and control strategies.     

Leishmaniases in Maghreb: An endemic neglected disease

Maghreb is known to be one of the most endemic areas of leishmaniases where both visceral and cutaneous forms are reported. Cutaneous leishmaniasis (CL) is older and has a higher prevalence than visceral one (VL). It is caused by four taxa (Leishmania (L.) major, L. infantum, L. tropica and L. killicki) which are responsible for a large clinical spectrum of lesions. Most transmission cycles of these taxa are known and many phlebotomine sandflies vectors and reservoir hosts are identified. The zoonotic transmission is well established for L. major. However, for L. infantum and L. killicki it needs more investigations to be proven. Regarding L. tropica, studies suggest it to be of both zoonotic and anthroponotic types. The isoenzymatic characterization of these four taxa showed a large enzymatic polymorphism varying from two zymodemes for L. major to 10 zymodemes for L. tropica. Cutaneous leishmaniasis is widely distributed and covers all bioclimatic stages with the coexistence of more than one taxon in the same foci.     

Phlebotomus papatasi and Meriones libycus as the vector and reservoir host of cutaneous leishmaniasis in Qomrood District, Qom Province, central Iran

ObjectiveTo determine the sand flies species responsible for most transmission of Leishmania major (L. major) to human, as well as to determine the main reservoir hosts of the disease.MethodsSand flies were collected using sticky papers and mounted in Puri's medium for species identification. Rodents were trapped by live Sherman traps. Both sand flies and rodents were subjected to molecular methods for detection of leishmanial parasite.ResultsPhlebotomus papatasi (P. papatasi) was the common species in outdoor and indoor resting places. Employing PCR technique only three specimens of 150 P. papatasi (2%) were found naturally infected by parasites with a band of 350 bp which is equal to the L. major parasite. Forty six rodents were captured by Sherman traps and identified. Microscopic investigation on blood smear of the animals for amastigote parasites revealed 1 (3.22%) infected Meriones libycus (M. libycus). Infection of this animal to L. major was confirmed by PCR against rDNA loci of the parasite.ConclusionsThis is the first molecular report of parasite infection of both vector (P. papatasi) and reservoir (M. libycus) to L. major in the region. The results indicated that P. papatasi was the primary vector of the disease and circulating the parasite between human and reservoirs and M. libycus was the most important host reservoir for maintenance of the parasite source in the area.     

Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province,Iran

ObjectiveTo assess molecular characterization, distribution, seasonal activities of sandfly species and Leishmania parasites infecting them for this zoonotic cutaneous leishmaniasis focus.MethodsThe collections were carried out in 2009–2011 using CDC traps, Sticky Papers and manual aspirator in and around the villages in Abarkouh district. Individual sandflies were characterized by PCR amplification and sequencing of fragments of their mitochondrial cytochrome b gene. Leishmania parasite infections within sandflies were performed by targeting Cyt b, ITS-rDNA, k-DNA and microsatellite genes.ResultsThe PCR assays detected only Leishmania major (L. major). All infections (30) were found in the abundant and widespread vector Phlebotomus papatasi (P. papatasi). Small numbers of other sandfly species were also screened for infections, but none was found. Sergentomyia sintoni and P. papatasi were the predominant members in all locations of this district and in all habitats throughout the trapping season. Only five other sandfly species were found, namely Phlebotomus ansari, Phlebotomus caucasicus, Phlebotomus sergenti, Sergentomyia dentata and Sergentomyia merviney.ConclusionsIn the current survey, the only infections detected are of L. major in females of P. papatasi (30 out of 190). The rates of infection of P. papatasi by L. major are not significantly different in compare with other locations in Iran with no diversity of parasite strains. Zoonotic cutaneous leishmaniasis may have emerged only recently in Abarkouh district, and the reason may well be the instability of the transmission cycles there.     

Experimental acquisition,development, and transmission of Leishmania tropica by Phlebotomus duboscqi

We report experimental infection and transmission of Leishmania tropica (Wright), by the blood-feeding sand fly Phlebotomus duboscqi (Neveu-Lemaire). Groups of laboratory-reared female sand flies that fed “naturally” on L. tropica-infected hamsters, or artificially, via membrane feeding device, on a suspension of L. tropica amastigotes, were dissected at progressive time points post-feeding. Acquisition, retention and development of L. tropica through procyclic, nectomonad, and leptomonad stages to the infective metacyclic promastigote stage, and anterior progression of the parasites from abdominal midgut bloodmeal to the thoracic midgut were demonstrated in both groups. Membrane feeding on the concentrated amastigote suspension led to metacyclic promastigote infections in 60% of sand flies, whereas only 3% of P. duboscqi that fed naturally on an infected hamster developed metacyclics. Sand flies from both groups re-fed on naïve hamsters, but despite infections in 25–50% of membrane-fed and 2–3.5% of naturally fed flies, no skin lesions developed in the hamsters. After four months of observation these animals were euthanized and necropsied. Screening of the organs and tissue by polymerase chain reaction (PCR) that targeted the small subunit RNA gene, amplified generic Leishmania DNA from liver, spleen, bone marrow, and blood, but only from hamsters bitten by membrane-infected P. duboscqi. These results are notable in demonstrating the ability of P. duboscqi, originating from Kenya, to acquire, retain, develop, and transmit a Turkish strain of L. tropica originally isolated from a human case of cutaneous leishmaniasis. This marks the first demonstration of complete development and transmission of L. tropica by a member of the Phlebotomus subgenus of sand flies.     

First report of natural infection in hedgehogs with Leishmania major, a possible reservoir of zoonotic cutaneous leishmaniasis in Algeria

We report here the first known cases of natural infection of hedgehogs with Leishmania major. Cutaneous leishmaniasis is an important public health problem in the area of M'sila, a semi-arid province in Algeria's northern Sahara, where two species of hedgehog live, Atelerix algirus and Paraechinus aethiopicus. The aim of this research was to survey Leishmania infection in these hedgehogs and evaluate whether they were reservoir hosts of Leishmania in an endemic zoonotic focus of leishmaniasis. Serological and molecular methods were used to determine the presence of Leishmania in 24 hedgehogs caught directly by hand and identified at species level as 19 A. algirus and 5 P. aethiopicus. Specific anti-Leishmania antibodies were detected in 29.2% of individuals by Western blot and in 26.3% by ELISA. The real-time PCR performed in spleen, ear and blood samples detected Leishmania spp. DNA in 12.5% of the individuals, one A. algirus and two P. aethiopicus. Three skin and two spleen samples of these animals were found to be parasitized and were identified by molecular test as L. major. Considering our results, it is suggested that hedgehogs have a potential epidemiological role as reservoir hosts of L. major.     

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, Ségou, 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 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.¹Epidemiological 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.¹³In 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. {"type":"entrez-nucleotide","attrs":{"text":"J04654","term_id":"340604","term_text":"J04654"}}J04654), L. infantum (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF188701","term_id":"11464594","term_text":"AF188701"}}AF188701), L. tropica (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z32841","term_id":"483434","term_text":"Z32841"}}Z32841), and L. donovani (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF167718","term_id":"11990503","term_text":"AF167718"}}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.Open in a separate windowFigure 1.L. major is the only parasite species identified by PCR in clinically suspected cases of CL in Mali. Positive control lanes + L. major and + L. donovani contain 650-bp and 800-bp PCR products from samples containing L. major and L. donovani DNA. Negative control lane (NC) shows PCR result from a sample processed in parallel to DNA extracted samples. Lanes 1–4 contain PCR products from four microscopically suspected samples of CL. M lane contains 100-bp DNA ladder.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.Open in a separate windowFigure 2.Geographic distribution of PCR-confirmed cases of CL in Mali. Color coding represents frequency of cases originating from each district.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.     

High infection frequency,low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran

Smears of suspected patients infected with zoonotic cutaneous leishmaniasis (ZCL) were stained and examined under a light microscopic observation. DNA of parasites within human ulcers was extracted directly from their smears. Nested PCR was used to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) of Lesihmania parasites in human from Turkemen Sahara located in the northeastern part of Iran. Based on RFLP method by digesting BsuRI restriction enzyme and more precisely sequencing of DNA ITS-rDNA was shown to be species-specific. The infection rates of Leishmania parasites were high with 154 (93.9%) infections out of 164 suspected patients using microscopic observations. Only from 128 suspected patients out of 164, ITS-rDNA fragments were amplified and 125 samples had enough DNA to digest BsuRI restriction enzyme and do DNA sequencing. The Nested PCR assays detected not only Leishmania major but also Leishmania turanica for the first time, another parasite of the great gerbil in human. The density of L. major was high but the diversity was low with only 2 haplotypes. The overall ratio of L. major (123 infections) to L. turanica (2 infections) was significantly higher (Chi-squared test: p < 0.05). Infections of L. turanica are not reported only and/or not known to cause human disease. Our analytical framework conveys a clear understanding of both L. major and L. turanica which can only be approved as causative agents of ZCL by more extensive sampling and followed by standardized molecular diagnosis.     

Leishmania tropica in Egypt: an undesirable import

Cutaneous as well as visceral leishmaniasis has been previously reported in Egypt. The former clinical manifestation is attributed to Leishmania major, the latter to L. infantum. In this study, L. tropica was isolated from an Egyptian labourer returning from Saudi Arabia. Amastigotes were detected by both Giemsa staining and indirect immunofluorescence using rabbit anti-gp63. Promastigotes from Schneider's medium were typed isoenzymatically as L. tropica. In view of the emerging threat of visceralization of L. tropica, the potential risk for its transmission in Egypt is discussed.     

Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is one of the most important vector‐borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR‐RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites’ growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania’ growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL.     

First report of natural infection of phlebotomines for Leishmania (Leishmania) chagasi captured in Ponta Porã, on the border between Brazil and Paraguay

ObjectiveVisceral leishmaniasis has been reported in all Brazilian regions and, in Mato Grosso do Sul State, the occurrence of cases has increased significantly. The objective of this study was to identify the natural infection of phlebotomines by Leishmania in Ponta Porã, a Brazilian county bordering Paraguay.MethodsBy using light CDC and Shannon traps, 185 sandfly females were captured, dissected and arranged in 107 pools subjected to DNA extraction and amplification by Polymerase chain reaction.ResultsFrom the samples subjected to amplification, the fragment of 120 bp characteristic of Leishmania sp was observed in one of the groups, which is composed by a female species of Evandromyia cortelezzii (Brèthes, 1923).ConclusionsThe minimum infection rate calculated for Ponta Porã was 0.54%, and the occurrence of flagellates and the confirmation of natural infection by Leishmania chagasi pose a serious concern for the transmission of visceral leishmaniasis in the region.