Cutaneous leishmaniasis caused by Leishmania tropica in Kenya

9 leishmanial strains, isolated from cutaneous papulonodular lesions on 3 patients, were characterized by cellulose acetate electrophoresis using 7 enzymes. The patterns obtained were indistinguishable from those of a Leishmania tropica reference strain and these 9 strains were similar to L. tropica in failing to infect mice. Although these 3 patients were Americans, their only potential exposure to sandflies was in Kenya, and thus they are believed to be the first cases of cutaneous leishmaniasis due to L. tropica in Kenya.     

Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods

A PCR fingerprinting approach, using single non-specific primers, as well as restriction and single-strand conformation polymorphism (SSCP) analyses of the amplified ribosomal internal transcribed spacer, were used to investigate genetic variability in the species Leishmania tropica. Twenty-nine strains of the 'L. tropica complex' from different Old World geographical areas were studied including 4 from Namibia, and 1 strain of L. killicki. All techniques revealed a high degree of genetic heterogeneity among the strains of L. tropica. The PCR fingerprinting displayed the highest discriminatory power, but can be applied only to cultured parasites. The internal transcribed spacer (ITS) region can be amplified directly from infected clinical samples and analysed subsequently. No strict correlation was discerned between the genetic variants and either the geographical origin of the strains or the clinical manifestations associated with human disease, except for the Namibian strains. Also, genetic variation did not correlate well with characterization by enzyme variant electrophoretic analysis. The strain of L. killicki always clustered together with the strains of L. tropica, suggesting it, probably, should not be considered a separate species of Leishmania. However, the 4 Namibian strains formed a distinct, statistically well-supported group closely related to but different from the other strains of L. tropica.     

Genome-wide SNP and microsatellite variation illuminate population-level epidemiology in the Leishmania donovani species complex

The species of the Leishmania donovani species complex cause visceral leishmaniasis, a debilitating infectious disease transmitted by sandflies. Understanding molecular changes associated with population structure in these parasites can help unravel their epidemiology and spread in humans. In this study, we used a panel of standard microsatellite loci and genome-wide SNPs to investigate population-level diversity in L. donovani strains recently isolated from a small geographic area spanning India, Bihar and Nepal, and compared their variation to that found in diverse strains of the L. donovani complex isolates from Europe, Africa and Asia. Microsatellites and SNPs could clearly resolve the phylogenetic relationships of the strains between continents, and microsatellite phylogenies indicated that certain older Indian strains were closely related to African strains. In the context of the anti-malaria spraying campaigns in the 1960s, this was consistent with a pattern of episodic population size contractions and clonal expansions in these parasites that was supported by population history simulations. In sharp contrast to the low resolution provided by microsatellites, SNPs retained a much more fine-scale resolution of population-level variability to the extent that they identified four different lineages from the same region one of which was more closely related to African and European strains than to Indian or Nepalese ones. Joining results of in vitro testing the antimonial drug sensitivity with the phylogenetic signals from the SNP data highlighted protein-level mutations revealing a distinct drug-resistant group of Nepalese and Indian L. donovani. This study demonstrates the power of genomic data for exploring parasite population structure. Furthermore, markers defining different genetic groups have been discovered that could potentially be applied to investigate drug resistance in clinical Leishmania strains.     

Leishmania in the Old World: 2. Heterogeneity among L. tropica zymodemes

Isoenzyme profiles of 27 stocks of Leishmania tropica from widely separated geographical areas were compared with those of reference strains of L. tropica and L. major using starch-gel electrophoresis of 13 enzymes (GPI, GD, ES, PGM, PEPD, NH, ASAT, ALAT, PK, MPI, 6PGD, SOD, MDH). 18 zymodemes were seen. L. tropica showed considerable intraspecific variation which did not correlate with its epidemiological uniformity. Isolates from cases of cutaneous and visceral leishmaniasis and leishmaniasis recidivans were identified as L. tropica. Only one isoenzyme band was held in common with the enzyme profile of the L. major reference strain thus supporting the status of L. tropica as a separate species.     

Canine and rodent leishmanial isolates from Egypt

Two leishmanial stocks isolated in Egypt, one from a dog and the other from Rattus norvegicus, were typed according to their excreted factor (EF) serotype and the electrophoretic mobility of their glucose phosphate isomerase. The canine isolate was indistinguishable from L. donovani (= L. infantum ) strains. The rat isolate appeared to be different from L. donovani, L. tropica and L. major.     

三种利什曼原虫和我国新疆皮肤利什曼原虫kDNA分子杂交实验研究

本研究自新疆克拉玛依地区皮肤利什曼病（CL）患者的皮肤病变组织内抽提微量的利什曼原虫kDNA，采用引物13A、13B进行PCR扩增，获120bP扩增区带（CL－PCR－AP），并转移到尼龙膜上，分别与地高辛（Digoxigenin－11－dUTP）标记的L．tropica、L．infantum、L．gerbillikDNA探针进行Southern印迹杂交。结果：病变组织内利什曼原虫kDNA的PCR扩增区带仅与LtropicakDNA探针有明显的杂交信号，而与L.infantum、L.gerbillikDNA探针杂交则呈阴性反应。结论：LtropicakDNA与新疆克拉玛依地区CL患者的CL－PCR－AP之间存在同源关系。     

A new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Kenya.

We have identified a new rural focus of cutaneous leishmaniasis caused by Leishmania tropica in Muruku sublocation, Salama location, Laikipia district, Rift Valley province, Kenya. Based on a few available case histories, previous reports of L. tropica in Kenya indicated a tentative geographical distribution. Recently 6 indigenous Kenyans from the new focus, who had never travelled outside Kenya, developed cutaneous lesions on the face and/or extremities found to contain Leishmania by culture and smear. Most of the patients manifested the typical 'urban' dry sore which grew slowly into a nodule measuring 2 x 1 cm to 9.5 x 3 cm, and after some months formed a central crust surrounded by small satellite papules. After treatment with Pentostam (sodium stibogluconate), about 40% of the sores failed to heal completely, either scarring centrally with fulminating papules at the edges and spreading peripherally, or healing but then recrudescing at the edge of the scar. Stationary-phase promastigotes from culture isolates were analysed by cellulose acetate electrophoresis. Isoenzyme profiles of 6 isolates were compared with those of World Health Organization reference strains using 12 enzyme loci; they were indistinguishable from those of 2 L. tropica reference strains. All 6 case sites lay within a radius of 4 km. Several other suspected cases from the same area are being investigated.     

Distinguishing Leishmania tropica and Leishmania major in the Middle East using the polymerase chain reaction with kinetoplast DNA-specific primers

This paper reports attempts to develop a sensitive and inexpensive procedure for rapid diagnosis of cutaneous leishmaniasis at the species level using skin scrapings from patients. The presence of 3 species (Leishmania major, L. tropica and L. infantum) in Israel and the West Bank demonstrates the need for a species-specific detection method in this region. The primer pair Uni21/Lmj4 was developed on the basis of an L. major minicircle sequence but it also amplified other 'Old World' species of Leishmania. Due to species-specific differences in the size of minicircles, these primers can be used in the polymerase chain reaction to answer diagnostic and epidemiological questions.     

Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method

The aim of this study was to develop a simple, low-cost method for the detection and species differentiation of Leishmania directly from clinical samples, for routine use in a parasitology laboratory. A total of 87 samples was used, including 60 peripheral blood, seven bone marrow and 17 skin lesion material samples, derived from Greek patients with visceral or cutaneous leishmaniasis, and three reference strains. PCR was performed using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene. Identification of the Leishmania species studied was achieved by digestion with a single restriction endonuclease (RFLP), single-strand conformational polymorphism (SSCP) and DNA sequencing of the PCR-generated fragments. Typing identified all visceral and one cutaneous leishmaniasis strains as L. infantum, twelve of the cutaneous leishmaniasis strains as L. tropica and four as L. major. The described PCR method proved efficient for the detection of pathogenic Leishmania species in various clinical samples, most importantly in peripheral blood samples. Furthermore, PCR followed by a simple RFLP using a single restriction endonuclease was capable of identifying all Leishmania species commonly encountered in Greece.     

Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, Northern Israel

Transmission of Leishmania tropica was studied in 2 adjacent foci in Israel where vector populations differ. Only Phlebotomus sergenti was found infected with L. tropica in the southern focus; P. arabicus was the main vector in the northern focus. Rock hyraxes (Procavia capensis) were incriminated as reservoir hosts in both foci. L. tropica strains from the northern focus isolated from sand flies, cutaneous leishmaniasis cases, and rock hyraxes were antigenically similar to L. major, and strains from the southern focus were typically L. tropica. Laboratory studies showed that P. arabicus is a competent vector of L. tropica, and P. sergenti is essentially refractory to L. tropica from the northern focus. Susceptibility of P. arabicus may be mediated by O glycoproteins on the luminal surface of its midgut. The 2 foci differ with respect to parasites and vectors, but increasing peridomestic rock hyrax populations are probably responsible for emergence of cutaneous leishmaniasis in both foci.     

Leishmania species, drug unresponsiveness and visceral leishmaniasis in Bihar, India

Sixteen isolates obtained, in January 1998-December 1999, from splenic aspirates from sodium stibogluconate-resistant cases of visceral leishmaniasis (VL; Indian kala-azar) and drawn from different districts of Bihar (India) were identified as Leishmania donovani. By isoenzyme analysis, all the strains were found identical to the WHO reference strain L. donovani MON-2 and differed from L. tropica MON-5. This study suggested that resistant cases of VL in Bihar were caused by L. donovani and not by L. tropica. No new strain responsible for drug unresponsiveness emerged during this period and other cause or causes of emergence of drug resistance should be sought. All the patients were cured with amphotericin B.     

Leishmania in the Old World: 3. The distribution of L. aethiopica zymodemes

Isoenzyme profiles of 28 stocks of Leishmania aethiopica were compared with those of reference strains of L. aethiopica, L. tropica and L. major using starch-gel electrophoresis of 13 enzymes (GPI, GD, ES, PGM, PEPD, NH, ASAT, ALAT, PK, MPI, 6PGD, SOD, MDH). 13 zymodemes were seen. L. aethiopica showed some infraspecific variation. Stocks from Phlebotomus longipes and Procavia habessinica were indistinguishable from those from man. Stocks from cases of diffuse cutaneous and cutaneous leishmaniasis were indistinguishable. Only one enzyme pattern was held in common with the L. tropica reference strain enzyme profile, and none with the L. major reference strain. The status of L. aethiopica as a separate species is supported.     

Application of the indirect fluorescent antibody test in serodiagnosis of cutaneous leishmaniasis in experimentally infected mice and naturally infected Rhombomys opimus

The indirect fluorescent antibody test (IFAT) was used to detect leishmanial antibodies in experimentally inoculated mice with promastigote forms of Leishmania major, L. tropica and L. donovani infantum and in naturally infected Rhombomys opimus captured from the area endemic for cutaneous leishmaniasis in Esfahan, Iran. In the mice inoculated with L. major, the leishmanial lesion appeared at the site of inoculation and the leishmanial antibody level was much higher than in mice inoculated with other strains of Leishmania and in which no lesion was observed up to the 22nd week after inoculation. In R. opimus, the microscopical examination of the two smears prepared from the ears of each gerbil (one by the ordinary and the other by the sand-paper method) showed about 41% to be parasitologically positive. However, in IFAT about 84% were serologically positive. There was a good correlation between the percentage of thickened ears and leishmanial antibody titres in the gerbils.This investigation indicated that IFAT is a suitable serological technique for finding the reservoir hosts of leishmaniasis and determining leishmanial infection among rodents.     

Comparative study of biological characteristics of the causal agents of zoonotic and anthroponotic cutaneous leishmaniasis in the USSR

A comparative study was made of the virulence of Leishmania tropica major and L. tropica minor, the causal agents of the zoonotic and anthroponotic forms of cutaneous leishmaniasis, from the USSR and their antigenic characteristics were compared with those of other Leishmania species (L. donovani, L. adleri, and promastigotes from Turkmenian reptiles). The virulence of strains of L. t. major was slightly higher than that of strains of L. t. minor. However, the difference in virulence gradually levelled out in successive multiple passages of the strains in hamsters. A comparative serological study of L. t. major and L. t. minor revealed clear antigenic differences between them. The differences between L. t. major, L. t. minor and L. donovani were shown to be equal in degree.     

Cutaneous leishmaniasis in Kenya: transmission of Leishmania major to man by the bite of a naturally infected Phlebotomus duboscqi

One leishmanial stock was isolated from a Phlebotomus duboscqi female captured in Baringo District, Kenya, and others from papular lesions that developed at sites where this sandfly had fed on a man. When characterized by cellulose acetate electrophoresis (eight enzymes examined), these isolates proved to be identical to known Leishmania major strains from man and a rodent (Arvicanthis sp.) and different from L. donovani and L. adleri, which also occur in Baringo. This is the first case of human cutaneous leishmaniasis caused by L. major reported from Kenya.     

A comparison of enzyme-linked immunosorbent assay and indirect fluorescent antibody test in the sero-diagnosis of cutaneous and visceral leishmaniasis in Iran.

ELISA AND IFAT have been applied to the sero-diagnosis of cutaneous and visceral leishmaniasis and the levels of leishmanial antibody detected by Leishmania donovani antigens in both tests have been compared. From the results it appears that ELISA is a little more sensitive than IFAT, but IFAT seems to be more specific in detecting leishmanial antibodies. In both tests reactions between leishmanial antigen and some other infections, such as malaria and typhoid, were observed. These non-specific reactions reduce the validity of both tests, especially ELISA, in the sero-diagnosis of cutaneous leishmaniasis but, in visceral leishmaniasis, the leishmanial antibody levels were high enough to be unaffected by non-specific reactions. In general, ELISA is as good as IFAT and more practical in the sero-diagnosis and mass screening surveys for kala-azar.     

Epidemiological and clinical features of cutaneous leishmaniases in Jenin District, Palestine, including characterisation of the causative agents in clinical samples

During 2002-2009, 466 cases of cutaneous leishmaniasis (CL) were reported from Jenin District, Palestine, affecting both genders. The average annual incidence was 23 cases per 100000 inhabitants, increasing with age in children. Most cases presented a single lesion, generally on the face. Diagnosis and species identification was done by applying internal transcribed spacer 1 (ITS1) RFLP analysis to 47 isolates, of which 44 (93.6%) were Leishmania tropica and 3 (6.4%) were L. major. RFLP analysis was also performed on 256 skin tissue scrapings spotted onto filter papers, showing that 138 (53.9%) were positive, of which 50.7% were infected with L. tropica, 17.4% with L. major and 2.9% with L. donovani s.l., and 29.0% could not be identified. This is the first report from Palestine on human CL caused by L. infantum. Nine of the strains of L. tropica were subjected to multilocus enzyme electrophoresis, six of which belonged to the zymodeme MON-137 and three to a new zymodeme (MON-307). This separation was corroborated by excreted factor serotyping. This observation modifies the classical epidemiological view of CL in Palestine. Jenin District is an active focus of CL caused by L. tropica, where Phlebotomus sergenti, the putative vector, is abundant. These data suggest that CL is a zoonotic infection, but an animal reservoir has not been found.     

Zoonotic cutaneous leishmaniasis in Saudi Arabia: lesions healing naturally in man followed by a second infection with the same zymodeme of Leishmania major

A patient with a previous history of an infection with Leishmania b. braziliensis contracted zoonotic cutaneous leishmaniasis (ZCL) in the Al-Hassa oasis, Eastern Province, Saudi Arabia. Five lesions healed spontaneously over a period of 40 weeks without treatment. A year after acquiring ZCL he became infected again in the same focus. Isolates of parasites at both episodes were identified as L. major, zymodeme LON-4. Compared with the first infection of ZCL, parasites were fewer in the lesions on the second occasion, the lesions were smaller and healing was quicker (10 weeks). This work and a previous report of patients with active lesions and leishmanial scars suggest that second infections of L. major are not uncommon in the oasis where no autochthonous infections of other species of Leishmania have yet been recorded in man and only one species of Phlebotomus (P. papatasi) is known.     

Genetic diversity evaluation on Portuguese Leishmania infantum strains by multilocus microsatellite typing

Leishmania infantum is the main etiological agent of zoonotic visceral leishmaniasis in the Mediterranean region, including Portugal, but, given its low isoenzyme diversity in this country, the population structure is poorly known. A set of 14 polymorphic microsatellite markers was studied on 136 Portuguese Leishmania strains isolated from different hosts, geographic regions and different clinical forms. A total of 108 different genotypes were found, which is a degree of genetic diversity comparable to other regions, even within zymodeme MON-1. A single most common genotype was detected in 1:5 of all strains, which, with a greater number of multi-strain genotypes found in the Lisbon Metropolitan Region, particularly for human strains, was suggestive of the occurrence of clonal transmission. In addition, a high re-infection rate was found among HIV+ patients. Model based analysis by STRUCTURE uncovered two main populations (populations A and B, composed of MON-1 and non-MON-1 strains, respectively), with great genetic diversity between them, and two MON-1 sub-populations (A1 and A2). High inbreeding coefficients were found in these populations, although strains with mixed ancestry were identified, suggesting that recombination also plays a role in the epidemiology of this species in Portugal. Some but limited geographical differentiation was observed, with groups of strains from the same regions clustering together, particularly those from canine origin.Our results show that L. infantum isolates from Portugal present microsatellite diversity comparable to other regions and that different transmission models play a role in its epidemiology, from clonal transmission to recombination. In addition, although Portugal is a small country, mobility of people and animals is high and Leishmania can be probably easily disseminated between infected hosts throughout the country, two instances of seemingly local restricted transmission were identified.     

Genetic diversity of Leishmania donovani/infantum complex in China through microsatellite analysis

The Leishmania strains from different epidemic areas in China were assessed for their genetic relationship. Twenty-nine strains of Leishmania infantum isolated from 1950 to 2001 were subjected to multilocus microsatellite typing (MLMT) using 14 highly polymorphic microsatellite markers. Twenty-two MLMT profiles were recognized among the 29 L. infantum strains, which differed from one another in 13 loci. Bayesian model-based and distance-based analysis of the data inferred two main populations in China. Sixteen strains belonged to one population, which also comprised previously characterized strains of L. infantum non-MON1 and Leishmania donovani. The parasites within this population are assignable to a distinct cluster that is clearly separable from the populations of L. donovani elsewhere, i.e. India, Sri Lanka and East Africa, and L. infantum non-MON1 from Europe. The remaining 13 Chinese strains grouped together with strains of L. infantum MON1 into another population, but formed a separate cluster which genetically differs from the populations of L. infantum MON1 from Europe, the Middle East, Central Asia and North Africa. The existence of distinct groups of L. infantum MON1 and non-MON1/L. donovani suggests that the extant parasites in China may have been restricted there, but not recently introduced from elsewhere.